LOCUS Exported 6766 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses Cre recombinase and Renilla luciferase from the EFS promoter and one U6-driven sgRNA targeting LacZ. AAV backbone.. ACCESSION . VERSION . KEYWORDS AAV:ITR-U6-sgRNA(LacZ)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6766) AUTHORS Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F TITLE CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. JOURNAL Cell. 2014 Sep 24. pii: S0092-8674(14)01163-5. doi: 10.1016/j.cell.2014.09.014. PUBMED 25263330 REFERENCE 2 (bases 1 to 6766) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6766 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..141 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 12..141 /label=AAV2 ITR promoter 156..396 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 156..176 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 327..346 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 426..501 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 562..773 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 787..1719 /codon_start=1 /product="luciferase from the anthozoan coelenterate Renilla reniformis (sea pansy)" /label=Rluc /translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ" CDS 1735..1791 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 1792..1812 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2839..2865 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" misc_feature 2893..3481 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2946..2966) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3364..3375) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3513..3989 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3712..3731) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4029..4169 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4029..4158 /label=AAV2 ITR rep_origin 4244..4699 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4331..4350) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4541..4562 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4716..4735) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4835..4857 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4895..4913) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4981..5085 /gene="bla" /label=AmpR promoter CDS 5086..5946 /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(5304..5323) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6117..6705 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6606..6625 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgaggg cctatttccc atgattcctt 181 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa 241 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 301 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt 361 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgtgcga atacgcccac 421 gcgatgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa 481 aaagtggcac cgagtcggtg ctttttttct agaggtacca ggtcttgaaa ggagtgggaa 541 ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg 601 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa 661 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 721 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggcgtacg 781 gccaccatga cttcgaaagt ttatgatcca gaacaaagga aacggatgat aactggtccg 841 cagtggtggg ccagatgtaa acaaatgaat gttcttgatt catttattaa ttattatgat 901 tcagaaaaac atgcagaaaa tgctgttatt tttttacatg gtaacgcggc ctcttcttat 961 ttatggcgac atgttgtgcc acatattgag ccagtagcgc ggtgtattat accagacctt 1021 attggtatgg gcaaatcagg caaatctggt aatggttctt ataggttact tgatcattac 1081 aaatatctta ctgcatggtt tgaacttctt aatttaccaa agaagatcat ttttgtcggc 1141 catgattggg gtgcttgttt ggcatttcat tatagctatg agcatcaaga taagatcaaa 1201 gcaatagttc acgctgaaag tgtagtagat gtgattgaat catgggatga atggcctgat 1261 attgaagaag atattgcgtt gatcaaatct gaagaaggag aaaaaatggt tttggagaat 1321 aacttcttcg tggaaaccat gttgccatca aaaatcatga gaaagttaga accagaagaa 1381 tttgcagcat atcttgaacc attcaaagag aaaggtgaag ttcgtcgtcc aacattatca 1441 tggcctcgtg aaatcccgtt agtaaaaggt ggtaaacctg acgttgtaca aattgttagg 1501 aattataatg cttatctacg tgcaagtgat gatttaccaa aaatgtttat tgaatcggac 1561 ccaggattct tttccaatgc tattgttgaa ggtgccaaga agtttcctaa tactgaattt 1621 gtcaaagtaa aaggtcttca tttttcgcaa gaagatgcac ctgatgaaat gggaaaatat 1681 atcaaatcgt tcgttgagcg agttctcaaa aatgaacaag ctagcggaag cggagccact 1741 aacttctccc tgttgaaaca agcaggggat gtcgaagaga atcccgggcc acccaagaag 1801 aagaggaagg tgtccaatct cctgactgtt caccagaacc tccctgcgct gccagtagat 1861 gccactagcg atgaggtcag gaaaaatctc atggatatgt ttagggatag acaggcgttt 1921 tctgaacaca cctggaaaat gctgcttagc gtgtgccgat cctgggcagc ctggtgtaag 1981 ctgaacaatc gcaaatggtt ccccgccgag ccggaggacg tgcgcgatta cctgctgtat 2041 ctccaggcaa gagggctggc tgtcaagact atccagcagc acttgggcca actgaatatg 2101 ctgcatcgac gcagcgggct cccccggcct agcgattcaa acgcagtctc ccttgttatg 2161 aggagaatta gaaaggaaaa cgtagatgcg ggtgagaggg ctaagcaggc tctcgctttt 2221 gagcggactg atttcgacca ggtcagatcc ctgatggaga acagcgatcg gtgccaggac 2281 atcaggaacc tcgcatttct gggaattgca tataacacac ttctgcgcat agctgagatc 2341 gcccggatca gagtgaaaga catcagtcga acggacggcg gccggatgct tattcatatt 2401 ggacgcacaa agacattggt cagcaccgct ggcgttgaaa aggccttgtc cctgggcgta 2461 acgaagctgg tggaaagatg gatctcagtg tccggcgtgg ctgacgaccc taataattac 2521 ttgttctgtc gagtgagaaa aaacggagtc gccgcgccct ctgccaccag ccaattgagt 2581 acacgggccc ttgaagggat ctttgaggca acccaccgac tcatatacgg agccaaggat 2641 gacagtggcc agaggtatct cgcctggtca ggtcattctg ctagggtggg ggccgcacga 2701 gacatggcgc gggcaggagt ctccatacca gagattatgc aagctggagg ttggacaaat 2761 gtgaacatcg ttatgaacta tatccgcaat cttgactctg aaaccggggc catggtgaga 2821 ctgctcgaag atggtgacta cccatacgat gttccagatt acgcttaaga attcgatatc 2881 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 2941 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 3001 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 3061 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 3121 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 3181 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 3241 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt 3301 ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 3361 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 3421 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 3481 catcgatacc gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag 3541 tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat 3601 taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg 3661 tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg 3721 aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc 3781 aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc 3841 tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct 3901 ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg 3961 cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga 4021 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 4081 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 4141 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca 4201 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc 4261 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 4321 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 4381 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 4441 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 4501 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 4561 ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt 4621 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 4681 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag 4741 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 4801 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 4861 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 4921 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 4981 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 5041 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 5101 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 5161 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 5221 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 5281 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 5341 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 5401 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 5461 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 5521 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 5581 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 5641 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 5701 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 5761 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 5821 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 5881 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 5941 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 6001 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 6061 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 6121 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 6181 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 6241 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 6301 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 6361 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 6421 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 6481 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 6541 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 6601 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 6661 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 6721 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt //