LOCUS       sequence-237575-        7647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral vector that expresses shRNA under the 
            mouse U6 promoter. A CMV-EGFP reporter cassette is included in the 
            vector to monitor expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7647)
  AUTHORS   Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja 
            J, Rooney DL, Ihrig MM, McManus MT, Gertler FB, Scott ML, Van Parijs
            L
  TITLE     A lentivirus-based system to functionally silence genes in primary 
            mammalian cells, stem cells and transgenic mice by RNA interference.
  JOURNAL   Nat Genet. 2003 Mar . 33(3):401-6.
  PUBMED    12590264
REFERENCE   2  (bases 1 to 7647)
  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: "Nat Genet. 
            2003 Mar . 33(3):401-6."
COMMENT     Sequence Label: pLL3.7
FEATURES             Location/Qualifiers
     source          1..7647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        65..368
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        369..572
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      611..620
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(617..619,620..622,623..1336)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(617..619,620..622,623..1333)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(662..683)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(923..942)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1270..1291
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1356..1389)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1445..2033
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1498..1518)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1916..1927)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2036..2052)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             2562..2742
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(2804..3392)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2884..2903)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3563..4354,4355..4423))
                     /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     4186..4205
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4424..4528)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4603..4622)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4794..5173
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5175..5373
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             5391..5571
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5618..5743
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6240..6473
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6658..6702
                     /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    7000..7117
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        7176..7488
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     7407..7427
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     7505..7521
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(7563..7596)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
       61 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      121 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      181 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      241 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      301 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      361 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      421 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      481 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      541 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc
      601 gctaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      661 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      721 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      781 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      841 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
      901 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
      961 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1021 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1081 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1141 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1201 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1261 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1321 cgagctgtac aagtaggaat tcgtcgaggg acctaataac ttcgtatagc atacattata
     1381 cgaagttata catgtttaag ggttccggtt ccactaggta caattcgata tcaagcttat
     1441 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     1501 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     1561 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     1621 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     1681 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     1741 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     1801 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     1861 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     1921 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     1981 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
     2041 ccgtcgacct cgatcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag
     2101 ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag
     2161 tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact
     2221 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc
     2281 ttgatctgtg gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag
     2341 ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc
     2401 aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc
     2461 tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag
     2521 catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc
     2581 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     2641 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     2701 gatccctcag acccttttag tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc
     2761 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2821 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2881 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2941 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3001 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3061 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3121 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3181 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3241 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3301 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3361 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3421 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3481 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3541 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3601 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3661 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3721 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3781 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3841 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3901 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3961 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4021 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4081 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4141 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4201 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4261 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4321 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4381 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4441 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4501 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     4561 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     4621 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     4681 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     4741 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
     4801 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     4861 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     4921 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     4981 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     5041 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     5101 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     5161 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     5221 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     5281 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     5341 taggcgtgta cggtgggagg tctatataag cagcgcgttt tgcctgtact gggtctctct
     5401 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     5461 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     5521 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
     5581 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
     5641 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
     5701 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
     5761 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
     5821 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
     5881 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     5941 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     6001 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     6061 taagaccacc gcacagcaag cggccggccg ctgatcttca gacctggagg aggagatatg
     6121 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     6181 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     6241 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     6301 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     6361 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     6421 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     6481 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     6541 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     6601 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     6661 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     6721 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     6781 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     6841 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     6901 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     6961 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     7021 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     7081 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     7141 agatccagtt tggttagtac cgggcccgct ctagagatcc gacgcgccat ctctaggccc
     7201 gcgccggccc cctcgcacag acttgtggga gaagctcggc tactcccctg ccccggttaa
     7261 tttgcatata atatttccta gtaactatag aggcttaatg tgcgataaaa gacagataat
     7321 ctgttctttt taatactagc tacattttac atgataggct tggatttcta taagagatac
     7381 aaatactaaa ttattatttt aaaaaacagc acaaaaggaa actcacccta actgtaaagt
     7441 aattgtgtgt tttgagacta taaatatccc ttggagaaaa gccttgttaa cgcgcggtga
     7501 ccctcgaggt cgacggtatc gataagctcg cttcacgaga ttccagcagg tcgagggacc
     7561 taataacttc gtatagcata cattatacga agttatatta agggttccaa gcttaagcgg
     7621 ccgcgtggat aaccgtatta ccgccat
//
