LOCUS       sequence-222203-        7518 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 7518)
  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 7518)
  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-shLacZ-1735
FEATURES             Location/Qualifiers
     source          1..7518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    346..463
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        512..1022
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     941..959
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1044..1643
                     /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(1044..1063)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1540..1560
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1659..2247
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1712..1732)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2130..2141)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2319..2552
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2630..2751
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2667..2686)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2721..2740
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2791..2926
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2946..2965)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2947..2965)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2975..2992)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2975..2991)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2984..3006)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3133..3588
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3220..3239)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3430..3451
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3614..3718
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3719..3787,3788..4579)
                     /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(3937..3956)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4750..5338
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5239..5258
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5492..5509
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5626..5647
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5662..5667,5668..5685,5686..5692)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5700..5716
                     /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     5705..5727
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5724..5740
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5724..5740
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5759..5779
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5761..5779
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5807..6033
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6034..6214
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6261..6386
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6879..7112
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7297..7341
                     /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"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaggtaccg gccaacgtga cctatcccat tactcgagta atgggatagg tcacgttggt
      301 ttttgaattc tagatcttga gacaaatggc agtattcatc cacaatttta aaagaaaagg
      361 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
      421 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
      481 cagcagagat ccactttggc gccggctcga gggggcccgg gttgcgcctt ttccaaggca
      541 gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg
      601 ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg
      661 ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct
      721 tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg
      781 cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata
      841 gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg
      901 ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc
      961 ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc
     1021 agggggatcc accggagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     1081 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     1141 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     1201 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgccg
     1261 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     1321 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     1381 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     1441 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     1501 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     1561 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     1621 tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa
     1681 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     1741 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     1801 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     1861 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     1921 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     1981 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     2041 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     2101 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     2161 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     2221 ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc
     2281 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     2341 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
     2401 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     2461 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     2521 acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat
     2581 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat
     2641 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2701 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     2761 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     2821 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     2881 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca
     2941 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     3001 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     3061 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     3121 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3181 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3241 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3301 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3361 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3421 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3481 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3541 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     3601 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3661 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3721 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3781 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3841 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3901 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3961 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4021 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4081 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4141 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4201 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4261 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4321 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4381 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     4441 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4501 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4561 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4621 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4681 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4741 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4801 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4861 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4921 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4981 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5041 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5101 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5161 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5221 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5281 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5341 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5401 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5461 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     5521 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     5581 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     5641 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     5701 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
     5761 aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca
     5821 atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag
     5881 aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga
     5941 aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat
     6001 attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga
     6061 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     6121 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     6181 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
     6241 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
     6301 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
     6361 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
     6421 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
     6481 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
     6541 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     6601 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     6661 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     6721 aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa
     6781 ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag
     6841 agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc
     6901 ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga
     6961 caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa
     7021 cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct
     7081 gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc
     7141 atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt
     7201 tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata
     7261 cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa
     7321 ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata
     7381 aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt
     7441 tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca
     7501 accccgaggg gacccaga
//
