LOCUS Exported 5987 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV TALE synthesis backbone with NN half repeat; fused to nuclear localization sequence linker and VP64 transcriptional activator effector domain. Synapsin promoter for neuronal expression. . ACCESSION . VERSION . KEYWORDS pAAV_hSyn_TALEBB(NN)-NLS-VP64_2A_GFP_WPRE_bGHpA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5987) AUTHORS Konermann S, Brigham MD, Trevino A, Hsu PD, Heidenreich M, Le Cong, Platt RJ, Scott DA, Church GM, Zhang F TITLE Optical control of mammalian endogenous transcription and epigenetic states. JOURNAL Nature. 2013 Jul 23. doi: 10.1038/nature12466. PUBMED 23877069 REFERENCE 2 (bases 1 to 5987) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5987 /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 181..628 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" CDS 651..677 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 1367..1387 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1412..1561 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 1583..1636 /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 1637..2353 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 1637..2350 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(1679..1700) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1940..1959) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2287..2308 /label=EGFP-C /note="EGFP, forward primer" misc_feature 2378..2966 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2431..2451) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2849..2860) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3008..3025) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3014..3221 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 3250..3390 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3250..3379 /label=AAV2 ITR rep_origin 3465..3920 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3552..3571) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3762..3783 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3937..3956) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4056..4078 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4116..4134) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4202..4306 /gene="bla" /label=AmpR promoter CDS 4307..5167 /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(4525..4544) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5338..5926 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5827..5846 /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 gcgttgtaca gtgtctagac tgcagagggc cctgcgtatg 181 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga 241 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga 301 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg 361 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc 421 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg 481 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg 541 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg 601 aggagtcgtg tcgtgcctga gagcgcagtc gagaaggatc cgccaccatg tacccatacg 661 atgttccaga ttacgctgta gatttgagaa ctttgggata ttcacagcag cagcaggaaa 721 agatcaagcc caaagtgagg tcgacagtcg cgcagcatca cgaagcgctg gtgggtcatg 781 ggtttacaca tgcccacatc gtagccttgt cgcagcaccc tgcagccctt ggcacggtcg 841 ccgtcaagta ccaggacatg attgcggcgt tgccggaagc cacacatgag gcgatcgtcg 901 gtgtggggaa acagtggagc ggagcccgag cgcttgaggc cctgttgacg gtcgcgggag 961 agctgagagg gcctcccctt cagctggaca cgggccagtt gctgaagatc gcgaagcggg 1021 gaggagtcac ggcggtcgag gcggtgcacg cgtggcgcaa tgcgctcacg ggagcacccc 1081 tcaacctgac agagacccgt acgggtctcg actcacgcct gagcaggtag tggctattgc 1141 atccaataac gggggcagac ccgcactgga gtcaatcgtg gcccagcttt cgaggccgga 1201 ccccgcgctg gccgcactca ctaatgatca tcttgtagcg ctggcctgcc tcggcggacg 1261 acccgccttg gatgcggtga agaaggggct cccgcacgcg cctgcattga ttaagcggac 1321 caacagaagg attcccgaga ggacatcaca tcgagtggca gctagcccca agaagaagag 1381 aaaggtggag gccagcggtt ccggacgggc tgacgcattg gacgattttg atctggatat 1441 gctgggaagt gacgccctcg atgattttga ccttgacatg cttggttcgg atgcccttga 1501 tgactttgac ctcgacatgc tcggcagtga cgcccttgat gatttcgacc tggacatgct 1561 gattaactct agaggcagtg gagagggcag aggaagtctg ctaacatgcg gtgacgtcga 1621 ggagaatcct ggcccagtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 1681 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 1741 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 1801 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc 1861 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 1921 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 1981 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 2041 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga 2101 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag 2161 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 2221 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 2281 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 2341 gctgtacaag taagaattcg atatcaagct tatcgataat caacctctgg attacaaaat 2401 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc 2461 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt 2521 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg 2581 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg 2641 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc 2701 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt 2761 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct 2821 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg 2881 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg 2941 gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgagc tagagctcgc 3001 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 3061 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 3121 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 3181 aagggggagg attgggaaga caatagcagg catgctgggg aggtaaccac gtgcggaccg 3241 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc 3301 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 3361 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc 3421 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg 3481 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 3541 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 3601 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 3661 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac 3721 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 3781 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat 3841 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 3901 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata 3961 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 4021 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 4081 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata 4141 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 4201 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 4261 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 4321 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 4381 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 4441 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 4501 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 4561 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 4621 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 4681 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 4741 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 4801 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 4861 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 4921 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 4981 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc 5041 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 5101 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 5161 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 5221 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 5281 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 5341 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 5401 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 5461 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 5521 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 5581 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 5641 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 5701 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 5761 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 5821 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 5881 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 5941 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt //