LOCUS       sequence-175335-        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   Firestein R, Bass AJ, Kim SY, Dunn IF, Silver SJ, Guney I, Freed E, 
            Ligon AH, Vena N, Ogino S, Chheda MG, Tamayo P, Finn S, Shrestha Y, 
            Boehm JS, Jain S, Bojarski E, Mermel C, Barretina J, Chan JA, 
            Baselga J, Tabernero J, Root DE, Fuchs CS, Loda M, Shivdasani RA, 
            Meyerson M, Hahn WC
  TITLE     CDK8 is a colorectal cancer oncogene that regulates beta-catenin 
            activity.
  JOURNAL   Nature. 2008 Sep 25. 455(7212):547-51.
  PUBMED    18794900
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: "Nature. 
            2008 Sep 25. 455(7212):547-51."
COMMENT     Sequence Label: pLKO.1.sh.beta-catenin.1248
FEATURES             Location/Qualifiers
     source          1..7083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..67
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    184..205
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(220..225,226..243,244..250)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    258..274
                     /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     263..285
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     282..298
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     282..298
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     317..337
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        319..337
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        365..591
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             592..772
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    819..944
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1437..1670
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1855..1899
                     /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        2197..2445
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2197..2217
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2368..2387
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2541..2658
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2707..3217
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3136..3154
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3239..3838
                     /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(3239..3258)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3735..3755
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3966..4199
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4271..4392
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4308..4327)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4362..4381
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4432..4567
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4587..4606)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4588..4606)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4616..4633)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4616..4632)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4625..4647)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4774..5229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4861..4880)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5071..5092
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5255..5359
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5360..5428,5429..6220)
                     /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(5578..5597)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6391..6979
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6880..6899
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
       61 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
      121 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
      181 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
      241 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      301 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc
      361 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa
      421 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac
      481 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa
      541 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc
      601 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
      661 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
      721 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc
      781 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc
      841 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt
      901 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga
      961 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat
     1021 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt
     1081 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag
     1141 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa
     1201 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa
     1261 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg
     1321 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta
     1381 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga
     1441 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg
     1501 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg
     1561 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc
     1621 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg
     1681 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat
     1741 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac
     1801 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat
     1861 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca
     1921 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga
     1981 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg
     2041 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa
     2101 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtatcga
     2161 tcacgagact agcctcgagc ggccgccccc ttcaccgagg gcctatttcc catgattcct
     2221 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     2281 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     2341 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     2401 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggagg tgctatctgt
     2461 ctgctctact cgagtagagc agacagatag cacctttttt aattctcgac ctcgagacaa
     2521 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     2581 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     2641 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggccgcgg
     2701 ctcgaggggg ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg
     2761 ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt
     2821 cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac
     2881 gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg
     2941 acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat
     3001 ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga
     3061 gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct
     3121 gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg
     3181 ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat
     3241 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     3301 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     3361 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     3421 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     3481 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     3541 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     3601 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     3661 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     3721 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     3781 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg
     3841 cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcggtacc
     3901 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     3961 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
     4021 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     4081 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     4141 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     4201 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
     4261 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4321 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4381 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc
     4441 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4501 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     4561 ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     4621 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4681 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     4741 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     4801 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     4861 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4921 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4981 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5041 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5101 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     5161 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     5221 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5281 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5341 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5401 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     5461 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     5521 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     5581 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5641 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5701 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5761 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5821 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5881 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     5941 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6001 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6061 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6121 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6181 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6241 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6301 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6361 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6421 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     6481 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     6541 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     6601 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     6661 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     6721 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     6781 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     6841 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     6901 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     6961 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     7021 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     7081 tac
//
