LOCUS Exported 7410 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV-mediated expression of Chronos-GFP under the EF1-alpha promoter, in floxed/reversed (Cre-dependent) manner. Using bGHpA signal.. ACCESSION . VERSION . KEYWORDS pAAV-EF1-alpha-FLEX-rc[Chronos-GFP] SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7410) AUTHORS Klapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK, Morimoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, Constantine-Paton M, Wong GK, Boyden ES TITLE Independent optical excitation of distinct neural populations. JOURNAL Nat Methods. 2014 Mar;11(3):338-46. doi: 10.1038/nmeth.2836. Epub 2014 Feb 9. PUBMED 24509633 REFERENCE 2 (bases 1 to 7410) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7410 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 55..184 /label=AAV2 ITR primer_bind complement(250..267) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(421..1009) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(501..520) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1180..2040) /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 1803..1822 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2041..2145) /gene="bla" /label=AmpR promoter rep_origin complement(2171..2626) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2308..2329) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2520..2539 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 2698..2827 /label=AAV2 ITR intron 4177..4380 /label=EF-1-alpha intron A /note="truncated intron upstream of the start codon of human EF-1-alpha" primer_bind 4337..4357 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind complement(4409..4442) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind complement(4505..4538) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(4555..5268) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(4597..4618) /label=EGFP-C /note="EGFP, forward primer" primer_bind 4946..4965 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5205..5226 /label=EGFP-N /note="EGFP, reverse primer" regulatory 6258..6267 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 6289..6322 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind 6393..6426 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 6459..7047 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6512..6532) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6930..6941) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 7088..7295 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" ORIGIN 1 tcctagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac 61 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc 121 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc 181 gcagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 241 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 301 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 361 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 421 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 481 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 541 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 601 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 661 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 721 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 781 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 841 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 901 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 961 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 1021 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 1081 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 1141 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 1201 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 1261 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 1321 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 1381 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 1441 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 1501 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 1561 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 1621 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 1681 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 1741 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 1801 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 1861 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 1921 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 1981 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 2041 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 2101 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 2161 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 2221 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 2281 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 2341 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 2401 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 2461 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 2521 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 2581 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 2641 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 2701 cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt 2761 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg 2821 ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc atgctctagg 2881 aagatcgtag gttaattaag cagatctcga ccaattctca tgtttgacag cttatcatcg 2941 atcagctttg gagctaagcc agcaatggta gagggaagat tctgcacgtc ccttccaggc 3001 ggcctccccg tcaccacccc ccccaacccg ccccgaccgg agctgagagt aattcataca 3061 aaaggactcg cccctgcctt ggggaatccc agggaccgtc gttaaactcc cactaacgta 3121 gaacccagag atcgctgcgt tcccgccccc tcacccgccc gctctcgtca tcactgaggt 3181 ggagaagagc atgcgtgagg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca 3241 gtccccgaga agttgggggg aggggtcggc aattgaaccg gccggtgcct agagaaagtg 3301 gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg 3361 gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc 3421 cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta 3481 tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg attcttgatc 3541 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt 3601 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt 3661 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat 3721 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc 3781 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca 3841 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc 3901 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg 3961 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc 4021 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac 4081 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt 4141 accgggcgcc gtccaggcac ctcgattagt tctcgatcga gcttttggag tacgtcgtct 4201 ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact 4261 gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt 4321 ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag 4381 gtgtcgtgaa aactctagga gaattcccat aacttcgtat aaagtatcct atacgaagtt 4441 atatcaaaat aggaagacca atgcttcacc atcgacccga attgccaagc atcaccatcg 4501 acccataact tcgtataatg tatgctatac gaagttatac tagctagctc attacttgta 4561 cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg 4621 atcgcgcttc tcgttggggt ctttgctcag ggcggactgg gtgctcaggt agtggttgtc 4681 gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg 4741 cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg 4801 cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag 4861 gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc 4921 gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt 4981 gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg 5041 gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca 5101 gggcacgggc agcttgccgg tggtgcaaat gaacttcagg gtcagcttgc cgtaggtggc 5161 atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc 5221 gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcactg ctactaccgg 5281 tgccgccact cctccctcct cctcctcgtc gacgaaggtc tcgatctcca tgttctcgcc 5341 ggccacgttg atggtggttg tcttccggat gtcgccgtgg atcaggatgt gctcgtggat 5401 cttcactctc aggtggtggc ccaggaagcc ccaagtgttc ttgctgatca gatccaggat 5461 caggtggccg attccgctgg tgtaaggtgt gatcaggccc agtccctcgt gtccggcgat 5521 gaacatcacg gggaacatca gccaagagca gaagaagacg taggccatga tcttgcaaat 5581 cttccggcag acgcctttgg gcagggtgtg gtagctctcg atatacacct tggcgatctg 5641 gaagaagcaa gtcacgccgt agaacaggcc gatcatgaag aacaggatct tcagggggcc 5701 ctttgtaaag gcggctgtga tcccccacac gatgttgccg atgtcggtca ccaggatggt 5761 catggtccgc ttgctgtact cttcgtgcag tccggtcagg ttgctcaggt ggatcaggat 5821 cacggggcaa gtcaggagcc acatgctgta ccgcagccaa atcacggctc ctccgttggt 5881 ctggtacact gtggctgggc tgtcgacctc gtggaacagc tcgatgaagc acttgacgag 5941 ctcgatcacg cagacgtaga cctcctccca gccggttgtg gccttgaagc tgtgccaacc 6001 gtagaagatg agctgcacga tagccacgat cacggtgaac cactggagtc ccactgcgat 6061 cttgtgctgc agctcggttc cgtggttgat gtgggggaag caaccgtgat cggctccggc 6121 tgttgtggcg ttagaggttt ctccgtgagc gtctgctgct ggtgtcacca ctgctgcggc 6181 gctcagcagg cctctaattg tggcttcggc gctaggcacg gctgagataa aggcgtgggt 6241 cattgtggcg gctgtttcca tggtggcggc gcggcctagg tacctagtat aacttcgtat 6301 aggatacttt atacgaagtt atcattggga ttcttcctat tttgatccaa gcatcaccat 6361 cgaccctcta gtccagatct caccatcgac ccataacttc gtatagcata cattatacga 6421 agttatgtcc ctcgaagagg ttcgcgaagc ttatcgataa tcaacctctg gattacaaaa 6481 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 6541 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 6601 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 6661 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 6721 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 6781 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 6841 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 6901 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 6961 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc 7021 ggatctccct ttgggccgcc tccccgcatc gataccgtcg acccgggcgg ccgctcataa 7081 agctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 7141 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 7201 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 7261 gaggattggg aagacaatag caggcatgct ggggatgcgg tccggactgt acaattgcat 7321 tcattttatg tttcaggttc agggggagat gtgggaggtt ttttaaagca agtaaaacct 7381 ctacaaatgt ggtaaaatcg ataaggatct //