LOCUS       Exported                8596 bp ds-DNA     linear   SYN 18-MAY-2021
DEFINITION  3rd generation lentiviral transfer vector. pPRIME cloning plasmid 
            with a CMV promoter controlling expression of dsRed and miR30-based 
            shRNA targeting firefly luciferase.
ACCESSION   .
VERSION     .
KEYWORDS    pPRIME-CMV-dsRed-FF3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8596)
  AUTHORS   Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ
  TITLE     A lentiviral microRNA-based system for single-copy polymerase 
            II-regulated RNA interference in mammalian cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2005 Sep 13. 102(37):13212-7.
  PUBMED    16141338
REFERENCE   2  (bases 1 to 8596)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 18, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8596
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        277..480
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             526..1203
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MDSSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(656..674)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(706..726)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     924..943
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1118..1141
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     ncRNA           1253..1374
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1401..1415
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1443..1569
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        1723..1825
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1826..2485
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1892..1911)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2615..2644
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     2669..2685
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2760..3348
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2813..2833)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3231..3242)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3351..3367)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             3877..4057
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(4119..4707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4199..4218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4878..5738)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5501..5520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5739..5843)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5918..5937)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6109..6488
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6490..6688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             6706..6886
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6933..7058
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7555..7788
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7973..8017
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    8315..8432
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        8569..276
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
       61 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactacc
      121 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      181 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
      241 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
      301 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
      361 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
      421 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
      481 ggtttagtga accgtcagat ccgctagcgc taccgggcct gcaggatgga ctcctccgag
      541 gacgtcatca aggagttcat gcgcttcaag gtgcgcatgg agggctccgt gaacggccac
      601 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
      661 ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc
      721 cagtacggct ccaaggtgta cgtgaagcac cccgccgaca tccccgacta caagaagctg
      781 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
      841 accgtgaccc aggactcctc cctgcaggac ggctccttca tctacaaggt gaagttcatc
      901 ggcgtgaact tcccctccga cggccccgta atgcagaaga agactatggg ctgggaggcc
      961 tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca caaggccctg
     1021 aagctgaagg acggcggcca ctacctggtg gagttcaagt ccatctacat ggccaagaag
     1081 cccgtgcagc tgcccggcta ctactacgtg gactccaagc tggacatcac ctcccacaac
     1141 gaggactaca ccatcgtgga gcagtacgag cgcgccgagg gccgccacca cctgttcctg
     1201 taggcggccg caagccttgt taagtgctcg cttcggcagc acatatacta tgttgaatga
     1261 ggcttcagta ctttacagaa tcgttgcctg cacatcttgg aaacacttgc tgggattact
     1321 tcttcaggtt aacccaacag aaggctcgag aaggtatatt gctgttgaca gtgagcgagc
     1381 tcccgtgaat tggaatccta gtgaagccac agatgtagga ttccaattca gcgggagcct
     1441 gcctactgcc tcggaattca aggggctact ttaggagcaa ttatcttgtt tactaaaact
     1501 gaataccttg ctatctcttt gatacatttt tacaaagctg aattaaaatg gtataaatta
     1561 aatcactttt ttcaattgga agactaatgc gtttaaacac gcggcgacgc gttcgaccga
     1621 ataaatacct gtgacggaag atcacttcgc agaataaata aatcctggtg tccctgttga
     1681 taccgggaag ccctgggcca acttttggcg aaaatgagac gttgatcggc acgtaagagg
     1741 ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg agttgtcgag
     1801 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
     1861 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
     1921 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
     1981 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
     2041 attacgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
     2101 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
     2161 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
     2221 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
     2281 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
     2341 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     2401 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     2461 cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc
     2521 tggttgctac gcctgaataa gtgataataa gcggatgaat ggcagaaatt cggatctcgc
     2581 gcgtttgggc ggtggctccc tgccacgcgg ctccgaacag aagctgatct ccgaagagga
     2641 tctgacatgt gtttaaactt aattaagtcg aggtcgacgg tatcgataag ctcgcttcac
     2701 gagatcatgt ttaagggttc cggttccact aggtacaatt cgatatcaag cttatcgata
     2761 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     2821 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     2881 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     2941 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     3001 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     3061 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     3121 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     3181 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3241 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     3301 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
     3361 gacctcgatc gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc
     3421 aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca
     3481 cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     3541 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat
     3601 ctgtggatct accacacaca aggctacttc cctgattggc agaactacac accagggcca
     3661 gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag
     3721 aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat
     3781 gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt
     3841 catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc
     3901 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     3961 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     4021 ctcagaccct tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa
     4081 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4141 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4201 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4261 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4321 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4381 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4441 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4501 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4561 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4621 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4681 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4741 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4801 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4861 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4921 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4981 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5041 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5101 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5161 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5221 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5281 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5341 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5401 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5461 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5521 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5581 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5641 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5701 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5761 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5821 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     5881 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     5941 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     6001 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     6061 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     6121 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     6181 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     6241 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     6301 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     6361 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     6421 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     6481 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     6541 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     6601 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     6661 gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta
     6721 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     6781 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     6841 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     6901 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     6961 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     7021 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     7081 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     7141 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     7201 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     7261 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     7321 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     7381 ccaccgcaca gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga
     7441 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     7501 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     7561 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     7621 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     7681 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     7741 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     7801 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     7861 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     7921 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     7981 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     8041 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     8101 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     8161 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     8221 tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc
     8281 aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg
     8341 ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat
     8401 tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc
     8461 cagtttggtt agtaccgggc ccgctctaga cgtattaccg ccatgcatta gttattaata
     8521 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     8581 tacggtaaat ggcccg
//
