LOCUS       Exported                7974 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses Cre-recombinase, a barcoded HDR template to introduce a 
            G13A mutation into the endogenous Kras locus, and a Kras-targeting 
            sgRNA to enhance HDR. .
ACCESSION   .
VERSION     .
KEYWORDS    AAV-KrasG13A/sgKras/Cre
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7974)
  AUTHORS   Winters IP, Chiou SH, Paulk NK, McFarland CD, Lalgudi PV, Ma RK, 
  TITLE     Multiplexed in vivo homology-directed repair and tumor barcoding 
            enables parallel quantification of Kras variant oncogenicity
  JOURNAL   Nature Communications 8: 2053 (2017)
REFERENCE   2  (bases 1 to 7974)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7974
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     regulatory      207..216
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        2241..2481
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2241..2261
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2412..2431
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2510..2585
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2673..2696)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3070..3089
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3141..3161
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3162..4190
                     /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(4694..4711)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4694..4710)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4703..4725)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4923..5377
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5009..5028)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5219..5240
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5394..5413)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5513..5535
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5573..5591)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5659..5763
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5764..6624
                     /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(5982..6001)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6795..7383
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7284..7303
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7537..7554
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7671..7692
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7707..7737
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7745..7761
                     /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     7750..7772
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7769..7785
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7769..7785
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
       61 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg
      121 gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtaaag atctgatatc
      181 atcgatcgcg atgcattaat taagcggccg ccatggcagt tcttttgtat ccatttgtct
      241 ctttatctgc aataatatat gttctacaag ggaaaggaga taaagtgtat atattaattt
      301 tatttcaaaa atgtttggaa aagcagacta ggaaacaaca gtgttgtatt cagggctgtt
      361 aactgtatac agtgccagca cttagtgcac ctgttgggag tgcagtgtat tataagcaag
      421 gcagaagtca cagaggttac tgagaaactc ttttatgttc tttacatttt ttttcttgcc
      481 aaaaatagtc tttatctctg aatttcattc aattttattt ccttgtacag aggtactgtt
      541 tttagtactc aaaggattat atgttaaaat tgatctgatc ctataccaaa tatttaaacc
      601 catttcccac agatagttta tagattaaaa tgccatcaaa tattatcttt atttgcttca
      661 ttgcctaaat atgtctttcc ccagcacagt gcagttttga caccagcttc ggcttcctat
      721 tttgttgccc tttattgcag aactgctctg atggctttaa aacagttctc tgcagttgtt
      781 ggctccaaca cagatgttct taggctacct aacttctaac ttttaatatc cagtcaacaa
      841 agaataccgc aagggtaggt gttgggatag ctgtcgacaa gctcatgcgg gtgtgtccac
      901 agggtatagc gtactatgca gaatatttgt actgagtgaa gtcatgatac attcctttga
      961 gagccattag ctgctacaaa acagtaatct ggctgtttag atcaacaagc taaatgatag
     1021 aagatgaaag tactggtttc catgtatttt tattaagtgt tgatgagaaa gttgtaagtg
     1081 acttacaggt tactctgtac atctgtagtc actgaattcg gaatatctta gagttttaca
     1141 cacctaggtg agtgttaaaa tattgataaa gtttttgata atcttgtgtg agacatgttc
     1201 taatttagtt gtattttatt atttttattg taaggcctgc tgaaaatgac tgagtataaa
     1261 ctagtagtcg ttggagctgg tgccgtgggg aaatcagcgc taacgataca gctaattcag
     1321 aatcactttg tggatgagta tgaccctacg atagaggtaa cgctgctcta cagtctgcgt
     1381 acgcttgtaa aggacggcag ccagccgctt tgaaaaagat atcattttta tatttattag
     1441 aaaattatat tgaaagttat ttcagttata tgtgatgtcc tttagttcca aggctttaaa
     1501 ctgggtgtta gggaaccata ggtgcaagaa agtccacttc tcatgagagc tcaccacaga
     1561 gaaagaaagt ccacttctca tgagagctta ccacagagag taaggtccaa gatgccatcc
     1621 gtgctttgta tttgcatttt gataaataaa tttcatgatt ttgtgagagt taagaatata
     1681 gctttgcctt tgatctattc ctgaaaggtt tgttgttgtt ttatgggttt gttgttattg
     1741 tgttgttgtt tggtgtcttt taggcagggt cttactatgc agccctgacc ggcctagaac
     1801 tcaactatgt agaccagact agctctgaat tctcagagat ccatctacct gcctctgctg
     1861 cccagatgct gcgattaaag atgtacacca ccatgtacag tgaaagttac tcttttgaaa
     1921 acactttatt gttatagatt acttggaatg ttgatgtgat gagaaaattt gattatacta
     1981 ggaaaataga atcacttgat ggttgagctt tttttttaag acttatttat tttatgtaag
     2041 tgagtacact gcttctctct tcagacacac cagaagaggg catcagatcc cattaagatg
     2101 gctgtgagcc accatgtggt tgctgggaat tgaactcagg acctctggaa aggcagtcag
     2161 tgctcttttt ttgtttaaag atttatttat ttttatgaat acagtgtcac tgtcttcata
     2221 cacacaagag ctcgagctgt gagggcctat ttcccatgat tccttcatat ttgcatatac
     2281 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     2341 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2401 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2461 tatatatctt gtggaaagga ccaaacaccg actgagtata aacttgtggg ttttagagct
     2521 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     2581 ggtgcttttt tgtttggcgc gcctatggct taattaacat ggcggccgca ttctaccggg
     2641 taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca
     2701 cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc
     2761 aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga
     2821 agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc
     2881 tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg
     2941 gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg
     3001 ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg
     3061 gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct
     3121 catctccggg acccgggatg cccaagaaga agaggaaggt gtccaattta ctgaccgtac
     3181 accaaaattt gcctgcatta ccggtcgatg caacgagtga tgaggttcgc aagaacctga
     3241 tggacatgtt cagggatcgc caggcgtttt ctgagcatac ctggaaaatg cttctgtccg
     3301 tttgccggtc gtgggcggca tggtgcaagt tgaataaccg gaaatggttt cccgcagaac
     3361 ctgaagatgt tcgcgattat cttctatatc ttcaggcgcg cggtctggca gtaaaaacta
     3421 tccagcaaca tttgggccag ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa
     3481 gtgacagcaa tgctgtttca ctggttatgc ggcggatccg aaaagaaaac gttgatgccg
     3541 gtgaacgtgc aaaacaggct ctagcgttcg aacgcactga tttcgaccag gttcgttcac
     3601 tcatggaaaa tagcgatcgc tgccaggata tacgtaatct ggcatttctg gggattgctt
     3661 ataacaccct gttacgtata gccgaaattg ccaggatcag ggttaaagat atctcacgta
     3721 ctgacggtgg gagaatgtta atccatattg gcagaacgaa aacgctggtt agcaccgcag
     3781 gtgtagagaa ggcacttagc ctgggggtaa ctaaactggt cgagcgatgg atttccgtct
     3841 ctggtgtagc tgatgatccg aataactacc tgttttgccg ggtcagaaaa aatggtgttg
     3901 ccgcgccatc tgccaccagc cagctatcaa ctcgcgccct ggaagggatt tttgaagcaa
     3961 ctcatcgatt gatttacggc gctaaggatg actctggtca gagatacctg gcctggtctg
     4021 gacacagtgc ccgtgtcgga gccgcgcgag atatggcccg cgctggagtt tcaataccgg
     4081 agatcatgca agctggtggc tggaccaatg taaatattgt catgaactat atccgtaacc
     4141 tggatagtga aacaggggca atggtgcgcc tgctggaaga tggcgattag ccatgtttaa
     4201 gggttcctct agtgtacacg cgttgtgcct tctagttgcc agccatctgt tgtttgcccc
     4261 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4321 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4381 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga acgcgtgata
     4441 tcagatctgt tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct
     4501 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc
     4561 aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
     4621 agagggagtg gccaaagcgc gcagctgcct gcaggtcgac tctagaggat ccccgggtac
     4681 cgagctcgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4741 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4801 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc
     4861 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     4921 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4981 cgctacactt gccagcgctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5041 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     5101 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     5161 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     5221 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     5281 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     5341 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     5401 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     5461 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5521 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     5581 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     5641 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5701 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5761 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5821 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5881 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5941 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6001 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6061 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6121 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6181 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6241 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6301 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6361 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6421 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6481 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6541 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6601 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6661 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6721 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6781 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6841 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6901 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6961 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7021 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7081 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     7141 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     7201 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7261 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7321 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7381 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7441 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     7501 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     7561 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     7621 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     7681 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     7741 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     7801 cttgcatgcc tgcaggcagc tgcgcgctcg aacttcatgc ctgccgacct tccccaggtc
     7861 acgatccgga cggcgggtga gttcacattt tarcagccgg acgtgcarac tccgctggtg
     7921 gtctaacgtc ggttaggtcc cttgaatcac gggacatatg ttggtgttgg aggt
//
