LOCUS       sequence-222226-        7519 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 7519)
  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 7519)
  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-shMBNL1-9155
FEATURES             Location/Qualifiers
     source          1..7519
                     /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    347..464
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        513..1023
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     942..960
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1045..1644
                     /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(1045..1064)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1541..1561
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1660..2248
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1713..1733)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2131..2142)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2320..2553
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2631..2752
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2668..2687)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2722..2741
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2792..2927
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2947..2966)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2948..2966)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2976..2993)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2976..2992)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2985..3007)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3134..3589
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3221..3240)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3431..3452
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3615..3719
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3720..3788,3789..4580)
                     /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(3938..3957)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4751..5339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5240..5259
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5493..5510
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5627..5648
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5663..5668,5669..5686,5687..5693)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5701..5717
                     /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     5706..5728
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5725..5741
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5725..5741
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5760..5780
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5762..5780
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5808..6034
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6035..6215
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6262..6387
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6880..7113
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7298..7342
                     /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 ggagataaat ggacgcaata acctcgaggt tattgcgtcc atttatctct
      301 tttttgaatt ctagatcttg agacaaatgg cagtattcat ccacaatttt aaaagaaaag
      361 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
      421 aaactaaaga attacaaaaa caaattacaa aaatttaaaa ttttcgggtt tattacaggg
      481 acagcagaga tccactttgg cgccggctcg agggggcccg ggttgcgcct tttccaaggc
      541 agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa acgcagcggc
      601 gccgaccctg ggtctcgcac attcttcacg tccgttcgca gcgtcacccg gatcttcgcc
      661 gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc
      721 ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac tagtaccctc
      781 gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc tgtggccaat
      841 agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt gcgggaggcg
      901 gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag
      961 cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga cctctctccc
     1021 cagggggatc caccggagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc
     1081 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     1141 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     1201 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcc
     1261 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     1321 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     1381 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     1441 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     1501 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     1561 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     1621 atgacccgca agcccggtgc ctgaacgcgt taagtcgaca atcaacctct ggattacaaa
     1681 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac
     1741 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc
     1801 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt
     1861 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc
     1921 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc
     1981 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg
     2041 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt
     2101 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc
     2161 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt
     2221 cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact tacaaggcag
     2281 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     2341 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     2401 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     2461 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     2521 gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca tgtcatctta
     2581 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     2641 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     2701 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     2761 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     2821 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     2881 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
     2941 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     3001 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     3061 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     3121 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3181 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3241 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3301 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3361 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3421 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3481 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3541 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     3601 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3661 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3721 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3781 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3841 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3901 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3961 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4021 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4081 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4141 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4201 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4261 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4321 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4381 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4441 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4501 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4561 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     4621 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4681 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4741 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4801 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4861 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4921 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4981 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5041 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5101 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5161 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5221 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5281 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5341 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5401 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5461 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5521 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     5581 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     5641 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     5701 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
     5761 caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc
     5821 aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga
     5881 gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg
     5941 aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga
     6001 tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg
     6061 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     6121 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     6181 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa
     6241 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg
     6301 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag
     6361 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg
     6421 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
     6481 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc
     6541 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga
     6601 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac
     6661 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag
     6721 caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga
     6781 attatataaa tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa
     6841 gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt
     6901 cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag
     6961 acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca
     7021 acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc
     7081 tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact
     7141 catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat
     7201 ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat
     7261 acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga
     7321 attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat
     7381 aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact
     7441 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc
     7501 aaccccgagg ggacccaga
//
