LOCUS       sequence-197795-        7500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral plasmid; This plasmid is the backbone for 
            the DECIPHER libraries (currently DISCONTINUED).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7500)
  AUTHORS   Frangou, G
  TITLE     DECIPHER library reagents
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7500)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro
FEATURES             Location/Qualifiers
     source          1..7500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             233..413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /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"
     promoter        1819..2067
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1819..1839
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1990..2009
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2194..2311
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2360..2759
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     regulatory      2766..2775
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2772..3482
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             3489..3542
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3549..4148
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3549..3568)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4045..4065
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4241..4474
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4546..4667
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4583..4602)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4637..4656
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4707..4842
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4875..4891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4875..4891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4888..4910)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4899..4915
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4923..4929,4930..4947,4948..4953))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4968..4989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5106..5123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5277..5865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5357..5376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6036..6827,6828..6896))
                     /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     6659..6678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6897..7001)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7069..7087
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7125..7147)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7247..7266
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7460..7482
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7474..7491
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7475..7491
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct
      241 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
      301 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
      361 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc
      421 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccact gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     1741 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg
     1801 atggccgccc ccttcaccga gggcctattt cccatgattc cttcatattt gcatatacga
     1861 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
     1921 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     1981 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     2041 tatatcttgt ggaaaggacg aaacaccgga gtcttctttt ttgaagacac ttcgtgcgtt
     2101 tggggtttcg gactgtagaa ctctgaacct ctcggtggtc gccgtatcat tagaattctc
     2161 gacctcgaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     2221 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     2281 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     2341 actttggccg ccgttcgaag gcctccgcgc cgggttttgg cgcctcccgc gggcgccccc
     2401 ctcctcacgg cgagcgctgc cacgtcagac gaagggcgca gcgagcgtcc tgatccttcc
     2461 gcccggacgc tcaggacagc ggcccgctgc tcataagact cggccttaga accccagtat
     2521 cagcagaagg acattttagg acgggacttg ggtgactcta gggcactggt tttctttcca
     2581 gagagcggaa caggcgagga aaagtagtcc cttctcggcg attctgcgga gggatctccg
     2641 tggggcggtg aacgccgatg attatataag gacgcgccgg gtgtggcaca gctagttccg
     2701 tcgcagccgg gatttgggtc gcggttcttg tttgtggatc gctgtgatcg tcacttggtc
     2761 tagacgccac catggtgtct aagggcgaag agctgattaa ggagaacatg cacatgaagc
     2821 tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca
     2881 agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct
     2941 tcgccttcga catcctggct accagcttca tgtacggcag cagaaccttc atcaaccaca
     3001 cccagggcat ccccgacttc tttaagcagt ccttccctga gggcttcaca tgggagagag
     3061 tcaccacata cgaggacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     3121 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga
     3181 tgcagaagaa aacactcggc tgggaggcca acaccgagat gctgtacccc gctgacggcg
     3241 gcctggaagg cagaagcgac atggccctga agctcgtggg cgggggccac ctgatctgca
     3301 acttcaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct
     3361 actatgtgga ccacagactg gaaagaatca aggaggccga caaagagacc tacgtcgagc
     3421 agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg cacaaactta
     3481 atggatccga gggcagagga agccttctaa catgcggtga cgtggaggag aatcccggcc
     3541 cttccgggat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     3601 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     3661 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     3721 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     3781 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     3841 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     3901 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     3961 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     4021 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     4081 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     4141 gtgcctgaat ctaggtcgac attctacttg gtacctttaa gaccaatgac ttacaaggca
     4201 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     4261 caacgaagat aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     4321 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     4381 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     4441 agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt
     4501 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca
     4561 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4621 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc
     4681 tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     4741 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     4801 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagacttt tgcagagacc
     4861 aaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4921 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4981 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5041 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5101 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5161 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5221 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5281 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5341 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5401 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5461 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5521 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5581 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5641 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5701 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5761 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5821 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5881 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5941 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6001 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6061 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6121 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6181 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6241 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6301 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6361 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6421 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6481 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6541 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6601 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6661 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6721 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6781 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6841 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6901 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6961 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7021 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     7081 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     7141 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     7201 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     7261 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     7321 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     7381 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     7441 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgccaagctg
//
