LOCUS Exported 6846 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION An AAV vector that expresses a nuclear-localized iRFP713 reporter and improved Cre recombinase. ACCESSION . VERSION . KEYWORDS pOTTC1442 - pAAV CMV-IE Nuc-iRFP-2A-iCre SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6846) AUTHORS Back S, Necarsulmer J, Whitaker LR, Coke LM, Koivula P, Heathward EJ, Fortuno LV, Zhang Y, Yeh CG, Baldwin HA, Spencer MD, Mejias-Aponte CA, Pickel J, Hoffman AF, Spivak CE, Lupica CR, Underhill SM, Amara SG, Domanskyi A, Anttila JE, Airavaara M, Hope BT, Hamra FK, Richie CT, Harvey BK TITLE Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats. JOURNAL Neuron. 2019 Feb 8. pii: S0896-6273(19)30062-5. doi: 10.1016/j.neuron.2019.01.035. PUBMED 30792150 REFERENCE 2 (bases 1 to 6846) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6846 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" enhancer 205..508 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 509..712 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 663..683 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 747..756 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 753..1700 /codon_start=1 /product="phytochrome-based near-infrared fluorescent protein, originally termed iRFP (Filonov et al., 2011)" /label=iRFP713 /note="name was changed to iRFP713 (Shcherbakova and Verkhusha, 2013)" /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE" CDS 1734..1754 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1758..1778 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1782..1802 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1833..1886 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 1899..1919 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" misc_feature 2973..3561 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3026..3046) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3444..3455) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3593..4069 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3792..3811) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4109..4249 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4109..4238 /label=AAV2 ITR rep_origin 4324..4779 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4411..4430) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4621..4642 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4796..4815) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4915..4937 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4975..4993) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5061..5165 /gene="bla" /label=AmpR promoter CDS 5166..6026 /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(5384..5403) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6197..6785 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6686..6705 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggccgcac gcgttagtta ttaatagtaa tcaattacgg ggtcattagt 181 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 241 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 421 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 481 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg 541 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 601 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt 661 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt 721 gaaccgtcag atccggtacc ggatccgcca ccatggctga aggatccgtc gccaggcagc 781 ctgacctctt gacctgcgac gatgagccga tccatatccc cggtgccatc caaccgcatg 841 gactgctgct cgccctcgcc gccgacatga cgatcgttgc cggcagcgac aaccttcccg 901 aactcaccgg actggcgatc ggcgccctga tcggccgctc tgcggccgat gtcttcgact 961 cggagacgca caaccgtctg acgatcgcct tggccgagcc cggggcggcc gtcggagcac 1021 cgatcactgt cggcttcacg atgcgaaagg acgcaggctt catcggctcc tggcatcgcc 1081 atgatcagct catcttcctc gagctcgagc ctccccagcg ggacgtcgcc gagccgcagg 1141 cgttcttccg ccgcaccaac agcgccatcc gccgcctgca ggccgccgaa accttggaaa 1201 gcgcctgcgc cgccgcggcg caagaggtgc ggaagattac cggcttcgat cgggtgatga 1261 tctatcgctt cgcctccgac ttcagcggcg aagtgatcgc agaggatcgg tgcgccgagg 1321 tcgagtcaaa actaggcctg cactatcctg cctcaaccgt gccggcgcag gcccgtcggc 1381 tctataccat caacccggta cggatcattc ccgatatcaa ttatcggccg gtgccggtca 1441 ccccagacct caatccggtc accgggcggc cgattgatct tagcttcgcc atcctgcgca 1501 gcgtctcgcc cgtccatctg gaattcatgc gcaacatagg catgcacggc acgatgtcga 1561 tctcgatttt gcgcggcgag cgactgtggg gattgatcgt ttgccatcac cgaacgccgt 1621 actacgtcga tctcgatggc cgccaagcct gcgagctagt cgcccaggtt ctggcctggc 1681 agatcggcgt gatggaagag ttgtacaagt ccggcctgcg cagccgcgcc gacccaaaaa 1741 agaagcgcaa ggtagacccc aagaagaagc gcaaggtaga tccaaaaaag aagagaaagg 1801 tactgtacaa gatgcattct agaggctccg gagagggcag gggaagtctt ctaacatgcg 1861 gggacgtgga ggaaaatccc gggcccatcg atatggtgcc caagaagaag aggaaagtct 1921 ccaacctgct gactgtgcac caaaacctgc ctgccctccc tgtggatgcc acctctgatg 1981 aagtcaggaa gaacctgatg gacatgttca gggacaggca ggccttctct gaacacacct 2041 ggaagatgct cctgtctgtg tgcagatcct gggctgcctg gtgcaagctg aacaacagga 2101 aatggttccc tgctgaacct gaggatgtga gggactacct cctgtacctg caagccagag 2161 gcctggctgt gaagaccatc caacagcacc tgggccagct caacatgctg cacaggagat 2221 ctggcctgcc tcgcccttct gactccaatg ctgtgtccct ggtgatgagg agaatcagaa 2281 aggagaatgt ggatgctggg gagagagcca agcaggccct ggcctttgaa cgcactgact 2341 ttgaccaagt cagatccctg atggagaact ctgacagatg ccaggacatc aggaacctgg 2401 ccttcctggg cattgcctac aacaccctgc tgcgcattgc cgaaattgcc agaatcagag 2461 tgaaggacat ctcccgcacc gatggtggga gaatgctgat ccacattggc aggaccaaga 2521 ccctggtgtc cacagctggt gtggagaagg ccctgtccct gggggttacc aagctggtgg 2581 agagatggat ctctgtgtct ggtgtggctg atgaccccaa caactacctg ttctgccggg 2641 tcagaaagaa tggtgtggct gccccttctg ccacctccca actgtccacc cgggccctgg 2701 aagggatctt tgaggccacc caccgcctga tctatggtgc caaggatgac tctgggcaga 2761 gatacctggc ctggtctggc cactctgcca gagtgggtgc tgccagggac atggccaggg 2821 ctggtgtgtc catccctgaa atcatgcagg ctggtggctg gaccaatgtg aacattgtga 2881 tgaactacat cagaaacctg gactctgaga ctggggccat ggtgaggctg ctcgaggatg 2941 gggactgaga attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg 3001 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 3061 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 3121 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 3181 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 3241 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 3301 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 3361 cggggaaatc atcgtccttt ccttggctgc tcgcctatgt tgccacctgg attctgcgcg 3421 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 3481 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct 3541 ccctttgggc cgcctccccg catcgatacc gagcgctgct cgagagatct acgggtggca 3601 tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac 3661 cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa 3721 tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag 3781 ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc 3841 aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt tgttgggatt 3901 ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac 3961 catattggcc aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc 4021 caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgattttgta 4081 ggtaaccacg tgcggaccga gcggccgcag gaacccctag tgatggagtt ggccactccc 4141 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc 4201 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg 4261 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat 4321 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4381 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg 4441 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 4501 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg 4561 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4621 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt 4681 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat 4741 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa 4801 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 4861 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 4921 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 4981 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 5041 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 5101 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 5161 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 5221 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 5281 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 5341 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 5401 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 5461 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 5521 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 5581 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 5641 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 5701 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 5761 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 5821 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 5881 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 5941 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 6001 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 6061 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 6121 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 6181 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 6241 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 6301 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 6361 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 6421 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 6481 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 6541 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 6601 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 6661 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 6721 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 6781 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 6841 acatgt //