LOCUS       sequence-200267-        7748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cre-recombinase, a barcoded HDR template to introduce a 
            G13S mutation into the endogenous Kras locus, and a Kras-targeting 
            sgRNA to enhance HDR. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7748)
  AUTHORS   Winters IP, Chiou SH, Paulk NK, McFarland CD, Lalgudi PV, Ma RK, 
            Lisowski L, Connolly AJ, Petrov DA, Kay MA, Winslow MM
  TITLE     Multiplexed in vivo homology-directed repair and tumor barcoding 
            enables parallel quantification of Kras variant oncogenicity.
  JOURNAL   Nat Commun. 2017 Dec 12;8(1):2053. doi: 10.1038/s41467-017-01519-y.
  PUBMED    29233960
REFERENCE   2  (bases 1 to 7748)
  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: "Nat 
            Commun."; date: "2017-12-12"; volume: "8(1):2053. doi"; pages: " 
            10.1038/s41467-017-01519-y"
COMMENT     Sequence Label: AAV-KrasG13S/sgKras/Cre
FEATURES             Location/Qualifiers
     source          1..7748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      126..135
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        2160..2408
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2160..2180
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2331..2350
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2429..2504
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2557..3056
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2592..2615)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2989..3008
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3060..3080
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3081..4109
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     primer_bind     complement(4613..4630)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4613..4629)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4622..4644)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4842..5296
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4928..4947)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5138..5159
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5313..5332)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5432..5454
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5492..5510)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5578..5682
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5683..5751,5752..6543)
                     /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(5901..5920)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6714..7302
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7203..7222
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7590..7611
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7626..7631,7632..7649,7650..7656)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7664..7680
                     /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     7669..7691
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7688..7704
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7688..7704
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gcggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt
       61 tcctggaggg gtggagtcgt gacgtaaaga tctgatatca tcgatcgcga tgcattaatt
      121 aagcggccgc catggcagtt cttttgtatc catttgtctc tttatctgca ataatatatg
      181 ttctacaagg gaaaggagat aaagtgtata tattaatttt atttcaaaaa tgtttggaaa
      241 agcagactag gaaacaacag tgttgtattc agggctgtta actgtataca gtgccagcac
      301 ttagtgcacc tgttgggagt gcagtgtatt ataagcaagg cagaagtcac agaggttact
      361 gagaaactct tttatgttct ttacattttt tttcttgcca aaaatagtct ttatctctga
      421 atttcattca attttatttc cttgtacaga ggtactgttt ttagtactca aaggattata
      481 tgttaaaatt gatctgatcc tataccaaat atttaaaccc atttcccaca gatagtttat
      541 agattaaaat gccatcaaat attatcttta tttgcttcat tgcctaaata tgtctttccc
      601 cagcacagtg cagttttgac accagcttcg gcttcctatt ttgttgccct ttattgcaga
      661 actgctctga tggctttaaa acagttctct gcagttgttg gctccaacac agatgttctt
      721 aggctaccta acttctaact tttaatatcc agtcaacaaa gaataccgca agggtaggtg
      781 ttgggatagc tgtcgacaag ctcatgcggg tgtgtccaca gggtatagcg tactatgcag
      841 aatatttgta ctgagtgaag tcatgataca ttcctttgag agccattagc tgctacaaaa
      901 cagtaatctg gctgtttaga tcaacaagct aaatgataga agatgaaagt actggtttcc
      961 atgtattttt attaagtgtt gatgagaaag ttgtaagtga cttacaggtt actctgtaca
     1021 tctgtagtca ctgaattcgg aatatcttag agttttacac acctaggtga gtgttaaaat
     1081 attgataaag tttttgataa tcttgtgtga gacatgttct aatttagttg tattttatta
     1141 tttttattgt aaggcctgct gaaaatgact gagtataaac tagtagtcgt tggagctggt
     1201 agcgtcggga aatccgcact aacgatacag ctaattcaga atcactttgt ggatgagtat
     1261 gaccctacga tagaggtaac gctgctctac agtctgcgta cgcttgtaaa ggacggcagc
     1321 cagccgcttt gaaaaagata tcatttttat atttattaga aaattatatt gaaagttatt
     1381 tcagttatat gtgatgtcct ttagttccaa ggctttaaac tgggtgttag ggaaccatag
     1441 gtgcaagaaa gtccacttct catgagagct caccacagag aaagaaagtc cacttctcat
     1501 gagagcttac cacagagagt aaggtccaag atgccatccg tgctttgtat ttgcattttg
     1561 ataaataaat ttcatgattt tgtgagagtt aagaatatag ctttgccttt gatctattcc
     1621 tgaaaggttt gttgttgttt tatgggtttg ttgttattgt gttgttgttt ggtgtctttt
     1681 aggcagggtc ttactatgca gccctgaccg gcctagaact caactatgta gaccagacta
     1741 gctctgaatt ctcagagatc catctacctg cctctgctgc ccagatgctg cgattaaaga
     1801 tgtacaccac catgtacagt gaaagttact cttttgaaaa cactttattg ttatagatta
     1861 cttggaatgt tgatgtgatg agaaaatttg attatactag gaaaatagaa tcacttgatg
     1921 gttgagcttt ttttttaaga cttatttatt ttatgtaagt gagtacactg cttctctctt
     1981 cagacacacc agaagagggc atcagatccc attaagatgg ctgtgagcca ccatgtggtt
     2041 gctgggaatt gaactcagga cctctggaaa ggcagtcagt gctctttttt tgtttaaaga
     2101 tttatttatt tttatgaata cagtgtcact gtcttcatac acacaagagc tcgagctgtg
     2161 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     2221 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     2281 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     2341 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     2401 caaacaccga ctgagtataa acttgtgggt tttagagcta gaaatagcaa gttaaaataa
     2461 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gtttggcgcg
     2521 cctatggctt aattaacatg gcggccgcat tctaccgggt aggggaggcg cttttcccaa
     2581 ggcagtctgg agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc
     2641 tctggcctcg cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg
     2701 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     2761 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     2821 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     2881 gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg
     2941 gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc
     3001 ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccggga cccgggatgc
     3061 ccaagaagaa gaggaaggtg tccaatttac tgaccgtaca ccaaaatttg cctgcattac
     3121 cggtcgatgc aacgagtgat gaggttcgca agaacctgat ggacatgttc agggatcgcc
     3181 aggcgttttc tgagcatacc tggaaaatgc ttctgtccgt ttgccggtcg tgggcggcat
     3241 ggtgcaagtt gaataaccgg aaatggtttc ccgcagaacc tgaagatgtt cgcgattatc
     3301 ttctatatct tcaggcgcgc ggtctggcag taaaaactat ccagcaacat ttgggccagc
     3361 taaacatgct tcatcgtcgg tccgggctgc cacgaccaag tgacagcaat gctgtttcac
     3421 tggttatgcg gcggatccga aaagaaaacg ttgatgccgg tgaacgtgca aaacaggctc
     3481 tagcgttcga acgcactgat ttcgaccagg ttcgttcact catggaaaat agcgatcgct
     3541 gccaggatat acgtaatctg gcatttctgg ggattgctta taacaccctg ttacgtatag
     3601 ccgaaattgc caggatcagg gttaaagata tctcacgtac tgacggtggg agaatgttaa
     3661 tccatattgg cagaacgaaa acgctggtta gcaccgcagg tgtagagaag gcacttagcc
     3721 tgggggtaac taaactggtc gagcgatgga tttccgtctc tggtgtagct gatgatccga
     3781 ataactacct gttttgccgg gtcagaaaaa atggtgttgc cgcgccatct gccaccagcc
     3841 agctatcaac tcgcgccctg gaagggattt ttgaagcaac tcatcgattg atttacggcg
     3901 ctaaggatga ctctggtcag agatacctgg cctggtctgg acacagtgcc cgtgtcggag
     3961 ccgcgcgaga tatggcccgc gctggagttt caataccgga gatcatgcaa gctggtggct
     4021 ggaccaatgt aaatattgtc atgaactata tccgtaacct ggatagtgaa acaggggcaa
     4081 tggtgcgcct gctggaagat ggcgattagc catgtttaag ggttcctcta gtgtacacgc
     4141 gttgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     4201 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     4261 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     4321 tgggaagaca atagcaggca tgctggggaa cgcgtgatat cagatctgtt acgtagataa
     4381 gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc
     4441 ctctctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac
     4501 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaaagcgcg
     4561 cagctgcctg caggtcgact ctagaggatc cccgggtacc gagctcgaat tcactggccg
     4621 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     4681 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4741 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     4801 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4861 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgctta
     4921 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4981 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5041 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5101 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5161 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     5221 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5281 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5341 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5401 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5461 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5521 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5581 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5641 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5701 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5761 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5821 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5881 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5941 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     6001 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6061 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6121 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6181 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6241 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6301 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6361 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6421 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6481 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6541 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6601 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6661 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6721 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6781 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6841 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6901 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6961 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     7021 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7081 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7141 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7201 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7261 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7321 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     7381 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     7441 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     7501 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     7561 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     7621 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     7681 tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct gcaggcagct
     7741 gcgcgctc
//
