LOCUS       sequence-222225-        7084 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 7084)
  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 7084)
  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-shMBNL1-3964
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    105..222
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        271..781
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     700..718
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             803..1402
                     /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(803..822)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1299..1319
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1530..1763
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1835..1956
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1872..1891)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1926..1945
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1996..2131
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2151..2170)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2152..2170)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2180..2197)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2180..2196)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2189..2211)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2338..2793
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2425..2444)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2635..2656
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2819..2923
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2924..2992,2993..3784)
                     /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(3142..3161)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3955..4543
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4444..4463
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4697..4714
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4831..4852
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4867..4872,4873..4890,4891..4897)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4905..4921
                     /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     4910..4932
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4929..4945
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4929..4945
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4964..4984
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4966..4984
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5012..5238
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5239..5419
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5466..5591
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6084..6317
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6502..6546
                     /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(6844..7084,1..8)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6844..6864
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7015..7034
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gaaacaccgg gccaaccaga tacccataat actcgagtat tatgggtatc tggttggctt
       61 tttgaattct cgacctcgag acaaatggca gtattcatcc acaattttaa aagaaaaggg
      121 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
      181 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
      241 agcagagatc cactttggcc gcggctcgag ggggttgggg ttgcgccttt tccaaggcag
      301 ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc
      361 cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc
      421 tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt
      481 gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc
      541 agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg tggccaatag
      601 cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc gggaggcggg
      661 gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc
      721 tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc tctctcccca
      781 gggggatcca ccggagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
      841 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
      901 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
      961 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     1021 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     1081 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     1141 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     1201 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     1261 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     1321 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     1381 gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag
     1441 cgcacgaccc catgcatcgg tacctttaag accaatgact tacaaggcag ctgtagatct
     1501 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     1561 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     1621 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     1681 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     1741 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata
     1801 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     1861 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     1921 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     1981 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2041 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     2101 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg
     2161 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     2221 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     2281 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     2341 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2401 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2461 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2521 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2581 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2641 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2701 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2761 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     2821 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2881 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2941 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     3001 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     3061 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     3121 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     3181 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     3241 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     3301 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3361 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3421 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3481 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3541 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3601 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     3661 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3721 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3781 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3841 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3901 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3961 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4021 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4081 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4141 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4201 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4261 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     4321 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4381 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4441 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4501 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4561 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4621 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4681 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     4741 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4801 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     4861 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     4921 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
     4981 aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct
     5041 tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca
     5101 tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt
     5161 ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc
     5221 ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     5281 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     5341 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     5401 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
     5461 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
     5521 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
     5581 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     5641 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     5701 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     5761 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     5821 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     5881 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc
     5941 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     6001 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     6061 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     6121 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     6181 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     6241 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     6301 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     6361 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     6421 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     6481 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     6541 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     6601 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     6661 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc
     6721 gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag atccattcga
     6781 ttagtgaacg gatctcgacg gtatcgatca cgagactagc ctcgagcggc cgcccccttc
     6841 accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     6901 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     6961 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     7021 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     7081 ggac
//
