LOCUS Exported 8446 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes Cas9 nickase and gRNA targeting CDKN2A Exon2. ACCESSION . VERSION . KEYWORDS pEN35-CDKN2A-Ex2-L SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8446) AUTHORS Zeng H, Jorapur A, Shain AH, Lang UE, Torres R, Zhang Y, McNeal AS, Botton T, Lin J, Donne M, Bastian IN, Yu R, North JP, Pincus L, Ruben BS, Joseph NM, Yeh I, Bastian BC, Judson RL TITLE Bi-allelic Loss of CDKN2A Initiates Melanoma Invasion via BRN2 Activation. JOURNAL Cancer Cell. 2018 Jul 9;34(1):56-68.e9. doi: 10.1016/j.ccell.2018.05.014. PUBMED 29990501 REFERENCE 2 (bases 1 to 8446) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8446 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" enhancer 452..737 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 739..1016 /label=chicken beta-actin promoter intron 1017..1245 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 1266..1292 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1296..1316 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1326..5426 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5430..5450 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(5487..5504) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5493..5700 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 5709..5849 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5709..5838 /label=AAV2 ITR rep_origin 5924..6379 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6011..6030) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6221..6242 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6396..6415) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6515..6537 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6575..6593) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6661..6765 /gene="bla" /label=AmpR promoter CDS 6766..7626 /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(6984..7003) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7797..8385 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8286..8305 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg tggcggggtc ggcgcagttg tttaagagct atgctggaaa cagcatagca 301 agtttaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 361 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc 421 aattctgcag acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc 481 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt 541 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 601 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 661 attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 721 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc 781 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 841 gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg 901 gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt 961 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag 1021 tcgctgcgac gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc 1081 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg 1141 ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta 1201 atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca 1261 ccatgtaccc atacgatgtt ccagattacg cttcgccgaa gaaaaagcgc aaggtcgaag 1321 cgtccgacaa gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg 1381 tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc 1441 ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg 1501 aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct 1561 gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca 1621 gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg 1681 gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa 1741 agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc 1801 acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg 1861 acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc 1921 ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca 1981 gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggca 2041 acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg 2101 aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg 2161 cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca 2221 tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct 2281 ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc 2341 ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg 2401 ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc 2461 tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc 2521 ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc 2581 acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga 2641 tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca 2701 gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg 2761 aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata 2821 agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg 2881 tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc 2941 tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga 3001 ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa 3061 tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa 3121 ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg 3181 tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg 3241 cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg 3301 gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc 3361 tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg 3421 acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc 3481 tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga 3541 cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg 3601 tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga 3661 gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc 3721 ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc 3781 gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc 3841 atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa 3901 gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga 3961 agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc 4021 tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac 4081 agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga 4141 acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt 4201 ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca 4261 actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa 4321 agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga 4381 agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca 4441 gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc 4501 ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt 4561 ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg 4621 tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga 4681 tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg 4741 cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg 4801 tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg 4861 actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta 4921 agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac 4981 tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca 5041 gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg 5101 aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag 5161 tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata 5221 agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag 5281 cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca 5341 aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga 5401 tcgacctgtc tcagctggga ggcgacagcc ccaagaagaa gagaaaggtg gaggccagct 5461 aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 5521 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 5581 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 5641 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga 5701 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 5761 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 5821 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca 5881 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc 5941 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 6001 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 6061 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 6121 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 6181 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 6241 ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt 6301 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 6361 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag 6421 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 6481 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 6541 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 6601 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 6661 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 6721 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 6781 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 6841 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 6901 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 6961 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 7021 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 7081 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 7141 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 7201 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 7261 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 7321 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 7381 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 7441 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca 7501 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 7561 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 7621 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 7681 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 7741 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 7801 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 7861 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 7921 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 7981 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 8041 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 8101 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 8161 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 8221 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 8281 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 8341 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 8401 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt //