LOCUS Exported 7193 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Labeling of inhibitory synapses (green fluorescence) in cre-expressing neurons. ACCESSION . VERSION . KEYWORDS AAV-EF1A-DIO-Gephyrin.FingR-eGFP-CCR5TC SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7193) AUTHORS Bensussen S, Shankar S, Ching KH, Zemel D, Ta TL, Mount RA, Shroff SN, Gritton HJ, Fabris P, Vanbenschoten H, Beck C, Man HY, Han X TITLE A Viral Toolbox of Genetically Encoded Fluorescent Synaptic Tags. JOURNAL iScience. 2020 Jun 30;23(7):101330. doi: 10.1016/j.isci.2020.101330. PUBMED 32674057 REFERENCE 2 (bases 1 to 7193) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7193 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" protein_bind 145..156 /label=ZF binding site /bound_moiety="CCR5TC zinc-finger domain" /note="target sequence for the CCR5TC zinc-finger domain from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 2008; Gross et al., 2013)" promoter 168..1346 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 399..1337 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1294..1314 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" primer_bind 1363..1382 /label=T7 /note="T7 promoter, forward primer" promoter 1363..1381 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1444..1477 /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 1528..1561 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(1576..1701) /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the rat zinc finger protein Kid-1 (Witzgall et al., 1994)" /label=KRAB-A /translation="VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASM" CDS complement(1750..2091) /codon_start=1 /product="zinc-finger domain from the CCR5-224 (+) zinc-finger nuclease that recognizes the left half-site target sequence in the human CCR5 gene (Perez et al., 2008; Gross et al., 2013)" /label=CCR5TC /translation="FQCRICMRNFSDRSNLSRHIRTHTGEKPFACDICGRKFAISSNLN SHTKIHTGSQKPFQCRICMRNFSRSDNLARHIRTHTGEKPFACDICGRKFATSGNLTRH TKIHLRGSQL" CDS complement(2137..2847) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELY" CDS complement(2137..2847) /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELY" primer_bind complement(2176..2197) /label=EGFP-C /note="EGFP, forward primer" primer_bind 2525..2544 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2784..2805 /label=EGFP-N /note="EGFP, reverse primer" CDS complement(2872..2895) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" protein_bind complement(3178..3211) /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(3262..3295) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 3320..3908 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3373..3393) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3791..3802) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3940..4416 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4139..4158) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4456..4596 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4456..4585 /label=AAV2 ITR rep_origin 4671..5126 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4758..4777) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4968..4989 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5143..5162) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5262..5284 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5322..5340) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5408..5512 /gene="bla" /label=AmpR promoter CDS 5513..6373 /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(5731..5750) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6544..7132 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7033..7052 /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 gcgtgtcatc ctcatcgaat tccgtgaggc tccggtgccc 181 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca 241 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact 301 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga 361 acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc 421 gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc 481 tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc 541 cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg 601 gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc 661 tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg 721 taaatgcggg ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg 781 gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga 841 gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc 901 cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg 961 aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg 1021 gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg 1081 cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct 1141 tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca 1201 ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat 1261 ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt 1321 tttttcttcc atttcaggtg tcgtgaggaa ttagcttggt actaatacga ctcactatag 1381 ggagacccaa gctggctagg taagcttggt accgagctcg gatcctctag agtcgactcc 1441 ggaataactt cgtataggat actttatacg aagttatgca gaatggtagc tggattgtag 1501 ctgctattag caatatgaaa cctcttaata acttcgtata gcatacatta tacgaagtta 1561 tggcgcgcct taagccatag aagcaagatt agaataattt tcaagcataa cttcacgata 1621 aagagaacgt tgagaaagat caagtttttt ccattcatca cgagtaaaaa gaacagcaac 1681 atcttcaaaa gtaacagaaa cagaaacttg agaagcacct tcacgttgtt caggagcgtc 1741 gacaatcgat agctggctgc cccgcaggtg aatctttgtg tgccgggtca gattgccgga 1801 ggtggcaaac tttctgccac agatatcaca ggcaaaaggt ttttcaccag tatgtgtccg 1861 gatgtgtctg gccaggttgt cgcttctgct aaagtttctc atacaaattc tgcactggaa 1921 aggtttctgg ctgccagtgt ggatcttggt gtggctgttc aggttgctgc tgatggcgaa 1981 cttccggccg caaatgtcgc aggcgaaggg cttttctccg gtgtgggttc tgatgtgcct 2041 gctcaggttg gaccggtcgc tgaagttccg catgcagatc cggcactgga atctagagcc 2101 agcccctgca cccgctccag cgccagctcc cctagagtac agctcgtcca tgccgagagt 2161 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc 2221 tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc 2281 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt 2341 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac 2401 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa 2461 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc 2521 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc 2581 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca 2641 ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt 2701 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga 2761 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc 2821 ggtgaacagc tcctcgccct tgctcacccc agatctactg gagccgctac ccttatcgtc 2881 gtcatccttg taatcggatc cggtgcggta gttgatggag atcgggatcg ggtcctggta 2941 gttccagtgg tcgatcgtga cggcgtacac cgtgatggta tagtcgacac caggtttcag 3001 gctgctgatg gtagcagtgg acttgctgcc aggcacggtg aattcctgga cagggctatt 3061 gccaccagtt tcaccgtagg tgatgcggta gtagcgaacc atgaccttgt acttgcccca 3121 gctgatctgg atgctggttg gtgatgcttc cttgacttcg agcatggtgg cgctagcata 3181 acttcgtata aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta 3241 ttctttagaa tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt 3301 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg 3361 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta 3421 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct 3481 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt 3541 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac 3601 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg 3661 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc 3721 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg 3781 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct 3841 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc 3901 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc 3961 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta 4021 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg 4081 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc 4141 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc 4201 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg 4261 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg 4321 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga 4381 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc 4441 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 4501 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 4561 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc 4621 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg 4681 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 4741 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 4801 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 4861 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg 4921 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 4981 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt 5041 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 5101 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc 5161 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 5221 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 5281 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 5341 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 5401 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 5461 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 5521 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 5581 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 5641 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 5701 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 5761 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 5821 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 5881 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 5941 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 6001 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 6061 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 6121 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 6181 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 6241 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 6301 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 6361 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 6421 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 6481 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 6541 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 6601 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 6661 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 6721 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 6781 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 6841 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 6901 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 6961 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 7021 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 7081 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 7141 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt //