LOCUS       Exported                7770 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses Cre-recombinase, a barcoded HDR template to introduce a 
            G12A mutation into the endogenous Kras locus, and a Kras-targeting 
            sgRNA to enhance HDR. .
ACCESSION   .
VERSION     .
KEYWORDS    AAV-KrasG12A/sgKras/Cre
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7770)
  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 7770)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7770
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      192..201
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        2226..2466
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2226..2246
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2397..2416
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2495..2570
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2658..2681)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3055..3074
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3126..3146
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3147..4175
                     /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"
     repeat_region   4476..4616
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4476..4605
                     /label=AAV2 ITR
     primer_bind     complement(4655..4672)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4655..4671)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4664..4686)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4884..5339
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4971..4990)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5181..5202
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5356..5375)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5475..5497
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5535..5553)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5621..5725
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5726..6586
                     /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(5944..5963)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6757..7345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7246..7265
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7499..7516
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7633..7654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7669..7699
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7707..7723
                     /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     7712..7734
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7731..7747
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7731..7747
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct ggaggggtgg agtcgtgacg taaagatctg atatcatcga tcgcgatgca
      181 ttaattaagc ggccgccatg gcagttcttt tgtatccatt tgtctcttta tctgcaataa
      241 tatatgttct acaagggaaa ggagataaag tgtatatatt aattttattt caaaaatgtt
      301 tggaaaagca gactaggaaa caacagtgtt gtattcaggg ctgttaactg tatacagtgc
      361 cagcacttag tgcacctgtt gggagtgcag tgtattataa gcaaggcaga agtcacagag
      421 gttactgaga aactctttta tgttctttac attttttttc ttgccaaaaa tagtctttat
      481 ctctgaattt cattcaattt tatttccttg tacagaggta ctgtttttag tactcaaagg
      541 attatatgtt aaaattgatc tgatcctata ccaaatattt aaacccattt cccacagata
      601 gtttatagat taaaatgcca tcaaatatta tctttatttg cttcattgcc taaatatgtc
      661 tttccccagc acagtgcagt tttgacacca gcttcggctt cctattttgt tgccctttat
      721 tgcagaactg ctctgatggc tttaaaacag ttctctgcag ttgttggctc caacacagat
      781 gttcttaggc tacctaactt ctaactttta atatccagtc aacaaagaat accgcaaggg
      841 taggtgttgg gatagctgtc gacaagctca tgcgggtgtg tccacagggt atagcgtact
      901 atgcagaata tttgtactga gtgaagtcat gatacattcc tttgagagcc attagctgct
      961 acaaaacagt aatctggctg tttagatcaa caagctaaat gatagaagat gaaagtactg
     1021 gtttccatgt atttttatta agtgttgatg agaaagttgt aagtgactta caggttactc
     1081 tgtacatctg tagtcactga attcggaata tcttagagtt ttacacacct aggtgagtgt
     1141 taaaatattg ataaagtttt tgataatctt gtgtgagaca tgttctaatt tagttgtatt
     1201 ttattatttt tattgtaagg cctgctgaaa atgactgagt ataaactagt agtcgttgga
     1261 gctgctggcg tgggcaagtc ggcccttacc atccagctaa ttcagaatca ctttgtggat
     1321 gagtatgacc ctacgataga ggtaacgctg ctctacagtc tgcgtacgct tgtaaaggac
     1381 ggcagccagc cgctttgaaa aagatatcat ttttatattt attagaaaat tatattgaaa
     1441 gttatttcag ttatatgtga tgtcctttag ttccaaggct ttaaactggg tgttagggaa
     1501 ccataggtgc aagaaagtcc acttctcatg agagctcacc acagagaaag aaagtccact
     1561 tctcatgaga gcttaccaca gagagtaagg tccaagatgc catccgtgct ttgtatttgc
     1621 attttgataa ataaatttca tgattttgtg agagttaaga atatagcttt gcctttgatc
     1681 tattcctgaa aggtttgttg ttgttttatg ggtttgttgt tattgtgttg ttgtttggtg
     1741 tcttttaggc agggtcttac tatgcagccc tgaccggcct agaactcaac tatgtagacc
     1801 agactagctc tgaattctca gagatccatc tacctgcctc tgctgcccag atgctgcgat
     1861 taaagatgta caccaccatg tacagtgaaa gttactcttt tgaaaacact ttattgttat
     1921 agattacttg gaatgttgat gtgatgagaa aatttgatta tactaggaaa atagaatcac
     1981 ttgatggttg agcttttttt ttaagactta tttattttat gtaagtgagt acactgcttc
     2041 tctcttcaga cacaccagaa gagggcatca gatcccatta agatggctgt gagccaccat
     2101 gtggttgctg ggaattgaac tcaggacctc tggaaaggca gtcagtgctc tttttttgtt
     2161 taaagattta tttattttta tgaatacagt gtcactgtct tcatacacac aagagctcga
     2221 gctgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     2281 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     2341 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     2401 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     2461 aaggaccaaa caccgactga gtataaactt gtgggtttta gagctagaaa tagcaagtta
     2521 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt
     2581 ggcgcgccta tggcttaatt aacatggcgg ccgcattcta ccgggtaggg gaggcgcttt
     2641 tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa
     2701 gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct
     2761 ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc
     2821 cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc
     2881 agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc
     2941 agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc
     3001 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
     3061 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggacccg
     3121 ggatgcccaa gaagaagagg aaggtgtcca atttactgac cgtacaccaa aatttgcctg
     3181 cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac atgttcaggg
     3241 atcgccaggc gttttctgag catacctgga aaatgcttct gtccgtttgc cggtcgtggg
     3301 cggcatggtg caagttgaat aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg
     3361 attatcttct atatcttcag gcgcgcggtc tggcagtaaa aactatccag caacatttgg
     3421 gccagctaaa catgcttcat cgtcggtccg ggctgccacg accaagtgac agcaatgctg
     3481 tttcactggt tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac
     3541 aggctctagc gttcgaacgc actgatttcg accaggttcg ttcactcatg gaaaatagcg
     3601 atcgctgcca ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac
     3661 gtatagccga aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa
     3721 tgttaatcca tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac
     3781 ttagcctggg ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg
     3841 atccgaataa ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca
     3901 ccagccagct atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt
     3961 acggcgctaa ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg
     4021 tcggagccgc gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg
     4081 gtggctggac caatgtaaat attgtcatga actatatccg taacctggat agtgaaacag
     4141 gggcaatggt gcgcctgctg gaagatggcg attagccatg tttaagggtt cctctagtgt
     4201 acacgcgttg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4261 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4321 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4381 gaggattggg aagacaatag caggcatgct ggggaacgcg tgatatcaga tctgttacgt
     4441 agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc
     4501 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4561 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggtcga
     4621 ctctagagga tccccgggta ccgagctcga attcactggc cgtcgtttta caacgtcgtg
     4681 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     4741 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     4801 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4861 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4921 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     4981 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5041 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     5101 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5161 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     5221 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     5281 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     5341 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     5401 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5461 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5521 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5581 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5641 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5701 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5761 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5821 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5881 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5941 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     6001 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6061 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6121 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6181 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6241 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6301 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6361 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6421 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6481 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6541 tagacagatc gctgagatag gtgcctcact gattaagtat tggtaactgt cagaccaagt
     6601 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6661 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6721 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6781 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6841 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6901 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6961 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7021 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7081 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7141 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7201 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7261 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7321 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7381 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7441 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7501 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     7561 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     7621 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     7681 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     7741 ctatgaccat gattacgcca agcttgcatg
//
