LOCUS Exported 5623 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION NHEJ donor for fusing a p2A-mCherry reporter to mouse Actb. EGFP driven by EF1a promoter and U6-driven sgRNAs targeting Actb. AAV backbone.. ACCESSION . VERSION . KEYWORDS AAV_Actb NHEJ donor_U6_sgRNA_EF1a_GFP_polyA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5623) AUTHORS Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, Shen X, Wei Y, Huang Z, Ying W, Wang Y, Nie YH, Zhang CC, Li S, Cheng L, Wang Q, Wu Y, Huang P, Sun Q, Shi L, Yang H TITLE Homology-mediated end joining-based targeted integration using CRISPR/Cas9. JOURNAL Cell Res. 2017 May 19. doi: 10.1038/cr.2017.76. PUBMED 28524166 REFERENCE 2 (bases 1 to 5623) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5623 /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 CDS 193..249 /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 250..960 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(395..413) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 663..682 /label=mCherry-F /note="mCherry, forward primer" promoter 990..1230 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 990..1010 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1161..1180 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 1260..1335 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 1393..1604 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 1617..1885 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" regulatory 1913..1922 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1919..2638 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1919..2635 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1964..1985) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2225..2244) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2572..2593 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2641..2865 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 2886..3026 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 2886..3015 /label=AAV2 ITR rep_origin 3101..3556 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3188..3207) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3398..3419 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3573..3592) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3692..3714 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3752..3770) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 3838..3942 /gene="bla" /label=AmpR promoter CDS 3943..4803 /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(4161..4180) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4974..5562 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5463..5482 /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 tgcggccatc gatgccaagc ttggtaccag tccgcctaga 181 agcacttgcg gcgccactaa cttctccctg ttgaaacaag caggggatgt cgaagagaat 241 cccgggccaa tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg 301 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag 361 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc 421 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac 481 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag 541 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc 601 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac 661 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc 721 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac 781 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc 841 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa 901 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtaa 961 agtccgccta gaagcacttg cggctcgagg agggcctatt tcccatgatt ccttcatatt 1021 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa 1081 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt 1141 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt 1201 tcttggcttt atatatcttg tggaaaggac gaaacaccga gtccgcctag aagcacttgg 1261 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg 1321 gcaccgagtc ggtgcttttt tgttttagag ctaggctagc aaggatctgc gatcgctccg 1381 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg 1441 tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg 1501 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg 1561 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acagctgaag cttcgagggg 1621 ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg ccgccatcca cgccggttga 1681 gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac tgcgtccgcc gtctaggtaa 1741 gtttaaagct caggtcgaga ccgggccttt gtccggcgct cccttggagc ctacctagac 1801 tcagccggct ctccacgctt tgcctgaccc tgcttgctca actctacgtc tttgtttcgt 1861 tttctgttct gcgccgttac agatccaagc tgtgaccggc gcctacaccg gtgccaccat 1921 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg 1981 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg 2041 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct 2101 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca 2161 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt 2221 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt 2281 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa 2341 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg 2401 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga 2461 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta 2521 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct 2581 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaga 2641 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 2701 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 2761 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 2821 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttgc ggccgcgtcg 2881 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac 2941 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag 3001 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct 3061 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca 3121 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 3181 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 3241 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 3301 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt 3361 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 3421 acaacactca accctatctc gggctattct tttgatttat aagggatttt gccgatttcg 3481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 3541 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 3601 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg 3661 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 3721 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt 3781 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 3841 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 3901 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 3961 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 4021 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 4081 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 4141 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 4201 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 4261 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 4321 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 4381 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 4441 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 4501 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 4561 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 4621 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 4681 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 4741 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 4801 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 4861 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 4921 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 4981 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 5041 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 5101 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 5161 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 5221 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 5281 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 5341 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 5401 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 5461 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 5521 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 5581 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt //