LOCUS       sequence-224206-        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-shMBNL3-9465
FEATURES             Location/Qualifiers
     source          1..7085
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    31..148
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        197..707
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     626..644
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             729..1328
                     /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(729..748)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1225..1245
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1456..1689
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1761..1882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1798..1817)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1852..1871
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1922..2057
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2077..2096)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2078..2096)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2106..2123)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2106..2122)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2115..2137)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2264..2719
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2351..2370)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2561..2582
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2745..2849
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2850..2918,2919..3710)
                     /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(3068..3087)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3881..4469
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4370..4389
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4623..4640
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4757..4778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4793..4798,4799..4816,4817..4823)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4831..4847
                     /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     4836..4858
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4855..4871
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4855..4871
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4890..4910
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4892..4910
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4938..5164
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5165..5345
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5392..5517
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6010..6243
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6428..6472
                     /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        6770..7018
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6770..6790
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6941..6960
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
       61 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
      121 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact
      181 ttggccgcgg ctcgaggggg ttggggttgc gccttttcca aggcagccct gggtttgcgc
      241 agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc
      301 gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc
      361 ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt
      421 gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc
      481 agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag
      541 ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag
      601 tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt
      661 cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg
      721 agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
      781 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
      841 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
      901 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
      961 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     1021 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     1081 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     1141 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     1201 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     1261 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     1321 gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg
     1381 catcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     1441 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     1501 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     1561 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     1621 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     1681 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     1741 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     1801 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     1861 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     1921 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     1981 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     2041 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     2101 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     2161 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     2221 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     2281 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2341 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2401 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2461 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2521 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2581 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2641 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2701 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2761 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2821 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     2881 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     2941 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3001 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3061 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3121 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3181 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3241 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3301 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3361 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3421 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3481 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3541 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3601 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3661 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3721 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3781 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3841 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3901 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3961 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4021 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4081 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4141 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4201 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4261 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4321 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4381 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4441 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4501 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4561 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4621 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     4681 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     4741 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     4801 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     4861 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
     4921 tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac
     4981 gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga
     5041 agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg
     5101 acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat
     5161 aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     5221 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     5281 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     5341 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
     5401 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
     5461 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
     5521 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
     5581 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
     5641 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
     5701 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
     5761 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
     5821 gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg
     5881 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
     5941 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
     6001 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
     6061 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
     6121 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
     6181 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
     6241 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
     6301 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
     6361 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
     6421 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
     6481 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
     6541 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
     6601 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg
     6661 aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc
     6721 tcgacggtat cgatcacgag actagcctcg agcggccgcc cccttcaccg agggcctatt
     6781 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     6841 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     6901 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     6961 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg
     7021 ccagccagca aattcaaata actcgagtta tttgaatttg ctggctggtt ttttgaattc
     7081 tcgac
//
