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" primer_bind complement(11..28) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(182..770) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(262..281) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(941..1801) /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 1564..1583 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1802..1906) /gene="bla" /label=AmpR promoter rep_origin complement(1932..2387) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2069..2090) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2281..2300 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 2459..2588 /label=AAV2 ITR intron 3938..4141 /label=EF-1-alpha intron A /note="truncated intron upstream of the start codon of human EF-1-alpha" primer_bind 4098..4118 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind complement(4170..4203) /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(4266..4299) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(4316..5029) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(4358..4379) /label=EGFP-C /note="EGFP, forward primer" primer_bind 4707..4726 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4966..4987 /label=EGFP-N /note="EGFP, reverse primer" regulatory 6019..6028 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 6050..6083 /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 6154..6187 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 6220..6808 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6273..6293) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6691..6702) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 6849..7056 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 7226..7355 /label=AAV2 ITR ORIGIN 1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt 2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 2641 gaagatcgta ggttaattaa gcagatctcg accaattctc atgtttgaca gcttatcatc 2701 gatcagcttt ggagctaagc cagcaatggt agagggaaga ttctgcacgt cccttccagg 2761 cggcctcccc gtcaccaccc cccccaaccc gccccgaccg gagctgagag taattcatac 2821 aaaaggactc gcccctgcct tggggaatcc cagggaccgt cgttaaactc ccactaacgt 2881 agaacccaga gatcgctgcg ttcccgcccc ctcacccgcc cgctctcgtc atcactgagg 2941 tggagaagag catgcgtgag gctccggtgc ccgtcagtgg gcagagcgca catcgcccac 3001 agtccccgag aagttggggg gaggggtcgg caattgaacc ggccggtgcc tagagaaagt 3061 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg 3121 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg 3181 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt 3241 atggcccttg cgtgccttga attacttcca cgcccctggc tgcagtacgt gattcttgat 3301 cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct 3361 tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg 3421 tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga 3481 tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg 3541 cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc 3601 acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct 3661 caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg 3721 gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc 3781 cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca 3841 cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag 3901 taccgggcgc cgtccaggca cctcgattag ttctcgatcg agcttttgga gtacgtcgtc 3961 tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac 4021 tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt 4081 tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca 4141 ggtgtcgtga aaactctagg agaattccca taacttcgta taaagtatcc tatacgaagt 4201 tatatcaaaa taggaagacc aatgcttcac catcgacccg aattgccaag catcaccatc 4261 gacccataac ttcgtataat gtatgctata cgaagttata ctagctagct cattacttgt 4321 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt 4381 gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt 4441 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct 4501 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct 4561 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca 4621 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt 4681 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg 4741 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt 4801 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc 4861 agggcacggg cagcttgccg gtggtgcaaa tgaacttcag ggtcagcttg ccgtaggtgg 4921 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct 4981 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcact gctactaccg 5041 gtgccgccac tcctccctcc tcctcctcgt cgacgaaggt ctcgatctcc atgttctcgc 5101 cggccacgtt gatggtggtt gtcttccgga tgtcgccgtg gatcaggatg tgctcgtgga 5161 tcttcactct caggtggtgg cccaggaagc cccaagtgtt cttgctgatc agatccagga 5221 tcaggtggcc gattccgctg gtgtaaggtg tgatcaggcc cagtccctcg tgtccggcga 5281 tgaacatcac ggggaacatc agccaagagc agaagaagac gtaggccatg atcttgcaaa 5341 tcttccggca gacgcctttg ggcagggtgt ggtagctctc gatatacacc ttggcgatct 5401 ggaagaagca agtcacgccg tagaacaggc cgatcatgaa gaacaggatc ttcagggggc 5461 cctttgtaaa ggcggctgtg atcccccaca cgatgttgcc gatgtcggtc accaggatgg 5521 tcatggtccg cttgctgtac tcttcgtgca gtccggtcag gttgctcagg tggatcagga 5581 tcacggggca agtcaggagc cacatgctgt accgcagcca aatcacggct cctccgttgg 5641 tctggtacac tgtggctggg ctgtcgacct cgtggaacag ctcgatgaag cacttgacga 5701 gctcgatcac gcagacgtag acctcctccc agccggttgt ggccttgaag ctgtgccaac 5761 cgtagaagat gagctgcacg atagccacga tcacggtgaa ccactggagt cccactgcga 5821 tcttgtgctg cagctcggtt ccgtggttga tgtgggggaa gcaaccgtga tcggctccgg 5881 ctgttgtggc gttagaggtt tctccgtgag cgtctgctgc tggtgtcacc actgctgcgg 5941 cgctcagcag gcctctaatt gtggcttcgg cgctaggcac ggctgagata aaggcgtggg 6001 tcattgtggc ggctgtttcc atggtggcgg cgcggcctag gtacctagta taacttcgta 6061 taggatactt tatacgaagt tatcattggg attcttccta ttttgatcca agcatcacca 6121 tcgaccctct agtccagatc tcaccatcga cccataactt cgtatagcat acattatacg 6181 aagttatgtc cctcgaagag gttcgcgaag cttatcgata atcaacctct ggattacaaa 6241 atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac 6301 gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc 6361 ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt 6421 ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc 6481 tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc 6541 gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg 6601 gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt 6661 ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc 6721 cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt 6781 cggatctccc tttgggccgc ctccccgcat cgataccgtc gacccgggcg gccgctcata 6841 aagctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 6901 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 6961 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 7021 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtccggactg tacaattgca 7081 ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc 7141 tctacaaatg tggtaaaatc gataaggatc ttcctagagc atggctacgt agataagtag 7201 catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct 7261 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt 7321 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca ttaatgaatc ggccaacgcg 7381 cggggagagg cggtttgcgt attgggcgct //