LOCUS       sequence-222223-        7085 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive lentiviral expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7085)
  AUTHORS   Li J, Choi PS, Chaffer CL, Labella K, Hwang JH, Giacomelli AO, Kim 
            JW, Ilic N, Doench JG, Ly SH, Dai C, Hagel K, Hong AL, Gjoerup O, 
            Goel S, Ge JY, Root DE, Zhao JJ, Brooks AN, Weinberg RA, Hahn WC
  TITLE     An alternative splicing switch in FLNB promotes the mesenchymal cell
            state in human breast cancer.
  JOURNAL   Elife. 2018 Jul 30;7. pii: 37184. doi: 10.7554/eLife.37184.
  PUBMED    30059005
REFERENCE   2  (bases 1 to 7085)
  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: "Elife."; 
            date: "2018-07-30"; volume: "7. pii: 37184. doi"; pages: " 
            10.7554/eLife.37184"
COMMENT     Sequence Label: pLKO.1-shMBNL3-0455
FEATURES             Location/Qualifiers
     source          1..7085
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    106..223
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        272..782
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     701..719
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             804..1403
                     /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(804..823)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1300..1320
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1531..1764
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1836..1957
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1873..1892)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1927..1946
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1997..2132
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2152..2171)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2153..2171)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2181..2198)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2181..2197)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2190..2212)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2339..2794
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2426..2445)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2636..2657
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2820..2924
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2925..2993,2994..3785)
                     /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     complement(3143..3162)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3956..4544
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4445..4464
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4698..4715
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4832..4853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4868..4873,4874..4891,4892..4898)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4906..4922
                     /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)."
     primer_bind     4911..4933
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4930..4946
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4930..4946
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4965..4985
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4967..4985
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5013..5239
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5240..5420
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5467..5592
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6085..6318
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6503..6547
                     /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        join(6845..7085,1..8)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6845..6865
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7016..7035
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gaaacaccgg catgattgaa gcgagtgata actcgagtta tcactcgctt caatcatgtt
       61 ttttgaattc tcgacctcga gacaaatggc agtattcatc cacaatttta aaagaaaagg
      121 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
      181 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
      241 cagcagagat ccactttggc cgcggctcga gggggttggg gttgcgcctt ttccaaggca
      301 gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg
      361 ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg
      421 ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct
      481 tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg
      541 cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata
      601 gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg
      661 ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc
      721 ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc
      781 agggggatcc accggagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
      841 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
      901 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
      961 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     1021 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     1081 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     1141 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     1201 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     1261 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     1321 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     1381 tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga
     1441 gcgcacgacc ccatgcatcg gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     1501 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     1561 aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc
     1621 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     1681 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     1741 agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat
     1801 aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg
     1861 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     1921 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc
     1981 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     2041 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     2101 ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt
     2161 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     2221 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2281 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     2341 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2401 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2461 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2521 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     2581 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     2641 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2701 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2761 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     2821 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2881 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2941 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3001 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3061 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3121 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3181 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3241 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3301 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3361 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3421 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3481 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3541 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3601 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3661 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3721 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3781 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3841 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3901 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3961 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4021 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4081 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4141 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4201 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4261 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4321 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4381 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4441 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4501 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4561 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4621 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4681 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     4741 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     4801 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     4861 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     4921 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
     4981 aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc
     5041 ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc
     5101 atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg
     5161 tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg
     5221 cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc
     5281 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     5341 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     5401 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
     5461 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
     5521 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
     5581 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
     5641 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
     5701 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
     5761 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     5821 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     5881 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat
     5941 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     6001 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     6061 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     6121 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     6181 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     6241 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     6301 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     6361 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     6421 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     6481 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     6541 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     6601 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     6661 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     6721 cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
     6781 attagtgaac ggatctcgac ggtatcgatc acgagactag cctcgagcgg ccgccccctt
     6841 caccgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     6901 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     6961 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     7021 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     7081 aggac
//
