LOCUS Exported 7267 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Cre-activated AAV expression of halorhodopsin 2.0 (eNpHR) fused to EYFP for optogenetic inhibition. ACCESSION . VERSION . KEYWORDS pAAV-double floxed-eNpHR-EYFP-WPRE-pA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7267) TITLE Cre-dependent expression of halorhodopsin REFERENCE 2 (bases 1 to 7267) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7267 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 231..1409 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 462..1400 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1357..1377 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 1440..1473 /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 1524..1557 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(1593..2306) /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(1635..1656) /label=EGFP-C /note="EGFP, forward primer" primer_bind 1984..2003 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2243..2264 /label=EGFP-N /note="EGFP, reverse primer" protein_bind complement(3252..3285) /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(3336..3369) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 3394..3982 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3447..3467) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3865..3876) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 4014..4490 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4213..4232) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4530..4670 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4530..4659 /label=AAV2 ITR rep_origin 4745..5200 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4832..4851) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5042..5063 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5217..5236) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5336..5358 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5396..5414) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5482..5586 /gene="bla" /label=AmpR promoter CDS 5587..6447 /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(5805..5824) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6618..7206 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7107..7126 /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga 181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg 241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg 301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt 361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg 421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt 481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct 541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga 601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct 661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt 721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc 781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg 841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac 901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc 961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag 1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct 1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg 1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga 1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc 1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg 1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa 1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa 1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc 1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc 1561 gcgcctcatt acacctcgtt ctcgtagcag aacttgtaca gctcgtccat gccgagagtg 1621 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct 1681 ttgctcaggg cggactggta gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg 1741 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg 1801 tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg 1861 ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag 1921 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg 1981 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg 2041 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg ggcgaagcac 2101 tgcaggccgt agccgaaggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg 2161 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac 2221 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg 2281 gtgaacagct cctcgccctt gctcacggcg gccgcatcat cagccggggt cccagaagca 2341 gatggaacat ccaaaatgct gccggacacg acagattcgt ttgacgtcag atagttcagg 2401 agcaagaatg cgaaaatgta ctttgccacg atatccagga aagaatatcc ccagctcgtc 2461 accccaacgg gcaacaccgc gataccctcg actccaagag cccacacaat tggatacccc 2521 agccacatca ctactgtcaa cagcttcagg gtattgaaca tgtcagcggt tcccgcggct 2581 ttggcgtcct gtgcccactc caccagcagg atatacaaga ccaccagaaa gcatgcacaa 2641 ctgatagcgt accagaacca tctcatgagg tgggaggagg tagtcagggc cgcggcaagg 2701 ccagtcacgc acatagcgat atcgaaagtg atagctgtga agagctttgt agcattgctt 2761 ccggccagga gacccagagc gaggagaatc atgggcgtgg aaagtgccca ggtgagatac 2821 cgtccccaca tggtgactac accatctacc tcttctcctc ccagcatcac tgagctgcct 2881 tctgcaaaat ggcccgctgg catttcaaga acggaaattg tcaggccgct cgccaatcca 2941 gtgtaggagg caatgctgac gacaggcaca aggatggttg acacagcaat aagttttgcc 3001 cgtggatcat cgagtcctcg ggtcataaaa acgaacagca gtatactcag tcctgcaagt 3061 gcgatgttga tatagagact gcttgcaagc aaagggtcgt tcagcacgaa ctcgaacaac 3121 tccctttggg taacctcggc ttgaagggcc acggcactct cggtcacggg aggcagggtc 3181 tctgtgtcct gaagcagaga gaacagagag acgacgagga gcaggggcgt acccctcatg 3241 gtggcgctag cataacttcg tataaagtat cctatacgaa gttatttgcc ttaacccaga 3301 aattatcact gttattcttt agaatggtgc aaagaataac ttcgtataat gtatgctata 3361 cgaagttatg aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt 3421 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct 3481 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat 3541 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg 3601 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag 3661 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc 3721 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg 3781 tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc 3841 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc 3901 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc 3961 tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc tacgggtggc 4021 atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca 4081 ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata 4141 atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta 4201 gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg 4261 caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat 4321 tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca 4381 ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc 4441 ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt 4501 aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc 4561 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 4621 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat 4681 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca 4741 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 4801 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc 4861 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 4921 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt 4981 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 5041 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat 5101 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa 5161 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca 5221 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 5281 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 5341 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 5401 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 5461 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 5521 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 5581 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 5641 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 5701 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 5761 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 5821 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 5881 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 5941 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 6001 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 6061 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 6121 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 6181 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 6241 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 6301 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 6361 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 6421 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 6481 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 6541 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 6601 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 6661 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 6721 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 6781 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 6841 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 6901 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 6961 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 7021 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 7081 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 7141 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 7201 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 7261 cacatgt //