LOCUS       sequence-152159-        7083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7083)
  AUTHORS   Guo W, Keckesova Z, Donaher JL, Shibue T, Tischler V, Reinhardt F, 
            Itzkovitz S, Noske A, Zurrer-Hardi U, Bell G, Tam WL, Mani SA, van 
            Oudenaarden A, Weinberg RA
  TITLE     Slug and Sox9 cooperatively determine the mammary stem cell state.
  JOURNAL   Cell. 2012 Mar 2;148(5):1015-28.
  PUBMED    22385965
REFERENCE   2  (bases 1 to 7083)
  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: "Cell."; 
            date: "2012-03-2"; volume: "148(5)"; pages: "1015-28"
COMMENT     Sequence Label: pLKO.1-sh-hSOX9-1
FEATURES             Location/Qualifiers
     source          1..7083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     240..260
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        288..514
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             515..695
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    742..867
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1360..1593
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1778..1822
                     /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        2120..2368
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2120..2140
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2291..2310
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2464..2581
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2630..3140
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3059..3077
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3162..3761
                     /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(3162..3181)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3658..3678
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3889..4122
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4194..4315
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4231..4250)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4285..4304
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4355..4490
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4510..4529)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4511..4529)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4539..4556)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4539..4555)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4548..4570)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4697..5152
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4784..4803)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4994..5015
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5178..5282
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5283..5351,5352..6143)
                     /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(5501..5520)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6314..6902
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6803..6822
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7056..7073
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
      241 caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc
      301 aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga
      361 gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg
      421 aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga
      481 tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg
      541 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
      601 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
      661 cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa
      721 gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg
      781 gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag
      841 aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg
      901 gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg
      961 gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc
     1021 tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga
     1081 tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac
     1141 accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag
     1201 caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga
     1261 attatataaa tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa
     1321 gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt
     1381 cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag
     1441 acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca
     1501 acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc
     1561 tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact
     1621 catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat
     1681 ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat
     1741 acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga
     1801 attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat
     1861 aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact
     1921 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc
     1981 aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag
     2041 agacagatcc attcgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg
     2101 agcggccgcc cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2161 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     2221 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2281 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2341 atatatcttg tggaaaggac gaaacaccgg gcatccttca atttctgtat actcgagtat
     2401 acagaaattg aaggatgctt tttaattctc gacctcgaga caaatggcag tattcatcca
     2461 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     2521 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     2581 tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg gggttggggt
     2641 tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct gggcgtggtt
     2701 ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg
     2761 tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct gctccgcccc
     2821 taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac
     2881 gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg cgccgaccgc
     2941 gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc ggccgggaag
     3001 gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt
     3061 gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc gttgaccgaa
     3121 tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag tacaagccca
     3181 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     3241 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     3301 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     3361 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     3421 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     3481 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     3541 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     3601 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     3661 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     3721 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc
     3781 agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga ccaatgactt
     3841 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     3901 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     3961 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     4021 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     4081 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc
     4141 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     4201 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4261 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4321 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4381 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4441 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     4501 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     4561 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4621 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4681 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4741 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4801 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4861 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     4921 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4981 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5041 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5101 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     5161 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5221 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5281 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5341 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5401 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5461 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5521 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5581 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5641 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5701 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5761 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5821 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5881 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5941 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6001 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6061 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6121 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6181 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6241 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6301 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6361 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6421 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6481 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6541 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6601 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6661 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6721 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6781 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6841 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6901 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6961 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     7021 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     7081 aag
//
