LOCUS       sequence-200269-        7666 bp    DNA     linear   UNA 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      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7666)
  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 7666)
  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..7666
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(58..75)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(58..74)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(67..89)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      287..742
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(374..393)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     584..605
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(759..778)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     878..900
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(938..956)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1024..1128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1129..1197,1198..1989)
                     /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
                     LIKYW"
     primer_bind     complement(1347..1366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2160..2748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2649..2668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3036..3057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3072..3077,3078..3095,3096..3102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3110..3126
                     /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     3115..3137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3134..3150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3134..3150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     regulatory      3320..3329
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        5354..5602
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5354..5374
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5525..5544
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5623..5698
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        5751..6250
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5786..5809)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6183..6202
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6254..6274
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6275..7303
                     /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"
ORIGIN
        1 gcgcgcagct gcctgcaggt cgactctaga ggatccccgg gtaccgagct cgaattcact
       61 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
      121 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
      181 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
      241 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
      301 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
      361 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
      421 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
      481 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
      541 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
      601 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
      661 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
      721 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
      781 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
      841 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
      901 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
      961 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     1021 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1081 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     1141 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     1201 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     1261 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     1321 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     1381 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     1441 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     1501 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     1561 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     1621 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     1681 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     1741 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     1801 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     1861 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     1921 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     1981 tattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     2041 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     2101 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2161 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2221 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2281 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     2341 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2401 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2461 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2521 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2581 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2641 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2701 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2761 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2821 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     2881 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     2941 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     3001 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     3061 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     3121 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc atgcctgcag
     3181 gcagctgcgc gctcgcggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
     3241 ccatcactag gggttcctgg aggggtggag tcgtgacgta aagatctgat atcatcgatc
     3301 gcgatgcatt aattaagcgg ccgccatggc agttcttttg tatccatttg tctctttatc
     3361 tgcaataata tatgttctac aagggaaagg agataaagtg tatatattaa ttttatttca
     3421 aaaatgtttg gaaaagcaga ctaggaaaca acagtgttgt attcagggct gttaactgta
     3481 tacagtgcca gcacttagtg cacctgttgg gagtgcagtg tattataagc aaggcagaag
     3541 tcacagaggt tactgagaaa ctcttttatg ttctttacat tttttttctt gccaaaaata
     3601 gtctttatct ctgaatttca ttcaatttta tttccttgta cagaggtact gtttttagta
     3661 ctcaaaggat tatatgttaa aattgatctg atcctatacc aaatatttaa acccatttcc
     3721 cacagatagt ttatagatta aaatgccatc aaatattatc tttatttgct tcattgccta
     3781 aatatgtctt tccccagcac agtgcagttt tgacaccagc ttcggcttcc tattttgttg
     3841 ccctttattg cagaactgct ctgatggctt taaaacagtt ctctgcagtt gttggctcca
     3901 acacagatgt tcttaggcta cctaacttct aacttttaat atccagtcaa caaagaatac
     3961 cgcaagggta ggtgttggga tagctgtcga caagctcatg cgggtgtgtc cacagggtat
     4021 agcgtactat gcagaatatt tgtactgagt gaagtcatga tacattcctt tgagagccat
     4081 tagctgctac aaaacagtaa tctggctgtt tagatcaaca agctaaatga tagaagatga
     4141 aagtactggt ttccatgtat ttttattaag tgttgatgag aaagttgtaa gtgacttaca
     4201 ggttactctg tacatctgta gtcactgaat tcggaatatc ttagagtttt acacacctag
     4261 gtgagtgtta aaatattgat aaagtttttg ataatcttgt gtgagacatg ttctaattta
     4321 gttgtatttt attattttta ttgtaaggcc tgctgaaaat gactgagtat aaactagtag
     4381 tcgttggagc tggtagcgtc gggaaatccg cactaacgat ccagctaatt cagaatcact
     4441 ttgtggatga gtatgaccct acgatagagg taacgctgct ctacagtctg cgtacgcttg
     4501 taaaggacgg cagccagccg ctttgaaaaa gatatcattt ttatatttat tagaaaatta
     4561 tattgaaagt tatttcagtt atatgtgatg tcctttagtt ccaaggcttt aaactgggtg
     4621 ttagggaacc ataggtgcaa gaaagtccac ttctcatgag agctcaccac agagaaagaa
     4681 agtccacttc tcatgagagc ttaccacaga gagtaaggtc caagatgcca tccgtgcttt
     4741 gtatttgcat tttgataaat aaatttcatg attttgtgag agttaagaat atagctttgc
     4801 ctttgatcta ttcctgaaag gtttgttgtt gttttatggg tttgttgtta ttgtgttgtt
     4861 gtttggtgtc ttttaggcag ggtcttacta tgcagccctg accggcctag aactcaacta
     4921 tgtagaccag actagctctg aattctcaga gatccatcta cctgcctctg ctgcccagat
     4981 gctgcgatta aagatgtaca ccaccatgta cagtgaaagt tactcttttg aaaacacttt
     5041 attgttatag attacttgga atgttgatgt gatgagaaaa tttgattata ctaggaaaat
     5101 agaatcactt gatggttgag cttttttttt aagacttatt tattttatgt aagtgagtac
     5161 actgcttctc tcttcagaca caccagaaga gggcatcaga tcccattaag atggctgtga
     5221 gccaccatgt ggttgctggg aattgaactc aggacctctg gaaaggcagt cagtgctctt
     5281 tttttgttta aagatttatt tatttttatg aatacagtgt cactgtcttc atacacacaa
     5341 gagctcgagc tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     5401 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     5461 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     5521 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     5581 cttgtggaaa ggaccaaaca ccgactgagt ataaacttgt gggttttaga gctagaaata
     5641 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     5701 ttttgtttgg cgcgcctatg gcttaattaa catggcggcc gcattctacc gggtagggga
     5761 ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg
     5821 ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc
     5881 tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc
     5941 ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag
     6001 tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg
     6061 ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg
     6121 gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc
     6181 ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc
     6241 gggacccggg atgcccaaga agaagaggaa ggtgtccaat ttactgaccg tacaccaaaa
     6301 tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc tgatggacat
     6361 gttcagggat cgccaggcgt tttctgagca tacctggaaa atgcttctgt ccgtttgccg
     6421 gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg tttcccgcag aacctgaaga
     6481 tgttcgcgat tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca
     6541 acatttgggc cagctaaaca tgcttcatcg tcggtccggg ctgccacgac caagtgacag
     6601 caatgctgtt tcactggtta tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg
     6661 tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac caggttcgtt cactcatgga
     6721 aaatagcgat cgctgccagg atatacgtaa tctggcattt ctggggattg cttataacac
     6781 cctgttacgt atagccgaaa ttgccaggat cagggttaaa gatatctcac gtactgacgg
     6841 tgggagaatg ttaatccata ttggcagaac gaaaacgctg gttagcaccg caggtgtaga
     6901 gaaggcactt agcctggggg taactaaact ggtcgagcga tggatttccg tctctggtgt
     6961 agctgatgat ccgaataact acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc
     7021 atctgccacc agccagctat caactcgcgc cctggaaggg atttttgaag caactcatcg
     7081 attgatttac ggcgctaagg atgactctgg tcagagatac ctggcctggt ctggacacag
     7141 tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga gtttcaatac cggagatcat
     7201 gcaagctggt ggctggacca atgtaaatat tgtcatgaac tatatccgta acctggatag
     7261 tgaaacaggg gcaatggtgc gcctgctgga agatggcgat tagccatgtt taagggttcc
     7321 tctagtgtac acgcgttgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     7381 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     7441 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     7501 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggaacgcgtg atatcagatc
     7561 tgttacgtag ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg
     7621 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccg
//
