LOCUS Exported 7782 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Express cre dependent TVA and SADB19 RG (biscistronic) as an AAV. ACCESSION . VERSION . KEYWORDS pAAV-EF1a-FLEX-T-RG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7782) AUTHORS Velez-Fort M, Bracey EF, Keshavarzi S, Rousseau CV, Cossell L, Lenzi SC, Strom M, Margrie TW TITLE A Circuit for Integration of Head- and Visual-Motion Signals in Layer 6 of Mouse Primary Visual Cortex. JOURNAL Neuron. 2018 Apr 4;98(1):179-191.e6. doi: 10.1016/j.neuron.2018.02.023. Epub 2018 Mar 15. PUBMED 29551490 REFERENCE 2 (bases 1 to 7782) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7782 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..141 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 12..141 /label=AAV2 ITR promoter 242..1419 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 473..1410 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1367..1387 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 1441..1450 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(1453..1486) /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(1549..1582) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(3173..3232) /codon_start=1 /product="2A peptide from equine rhinitis A virus polyprotein" /label=E2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="QCTNYALLKLAGDVESNPGP" protein_bind 3730..3763 /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 3834..3867 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 3909..4497 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3962..3982) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4380..4391) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 4529..5005 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4728..4747) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 5045..5185 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5045..5174 /label=AAV2 ITR rep_origin 5260..5715 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5347..5366) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5557..5578 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5732..5751) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5851..5873 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5911..5929) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5997..6101 /gene="bla" /label=AmpR promoter CDS 6102..6962 /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(6320..6339) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7133..7721 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7622..7641 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt 181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat 241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg 301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag 361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc 421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc 481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat 541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagcc 1441 gccaccatgg ccataacttc gtataaagta tcctatacga agttatatca aaataggaag 1501 accaatgctt caccatcgac ccgaattgcc aagcatcacc atcgacccat aacttcgtat 1561 aatgtatgct atacgaagtt atacttcaac tagttcacag tctggtctcg ccgccgctct 1621 tgtggctctc ccagctgctg atgatcttgc cgctctgggg ggtcacgctc acctctctgc 1681 cggtgcctct caggttgtgc tgggtgggct cgcttctgtt cactcttctg cagcaggtca 1741 tcaggaagat gatcagcatc agggcggtca gggcgccggc gctcagcagc acgtacttgc 1801 cccagttggg caggcccagg tccacgccgc tcacctggtt gtgcacgtcg ggcaggtgca 1861 cctccacgaa gtcctcggcc tcgtcgccgt ccttgaacac ggtgctgggg tcggccaggg 1921 ggtgcaccag ggggatcacg ctgctctcca gcagctccat gtgctgctgc agcaggctgc 1981 tctgcatctc ggggatcagc acgttgccgt cggggcccag gatgatgccg ttgaagaaca 2041 cgccgttcac gtgggggtgg catctgccgc ccactctcag gcagcccttg ctgggcagga 2101 tctcgttcca ggttctcacg ctcttgtagt gggcgtcggc ctccatcagg gtcttgttga 2161 agatggtgta ggccttgccg aagccgggca ccagctttct caggtggctc agtcttctga 2221 agctcacgct cttggtggtc atgatgctct ccagggcgtc caggcactcc tctctctttc 2281 tcaccagctc ctccaccacc aggtgctcga tctcgtcgct tctgaagtcg tgcaggttca 2341 ccagcttgtc gggggggcac cacttggtct cgttgctggt ctgcatgctc acccaggtgc 2401 cgtccatcag tctcaggccc agcacgccgc acagcttcag cttgcaggcg cccttcaggc 2461 tcttgtacag gcctctctcg tccacgaagc cgcaggtctc gctgcccttg ctggctctct 2521 tgcctctgct gttggtgaag atgtcgcagc tcatgcccag tctggggttc tcgggcatcc 2581 agatggtgta gtcgtggttg gtgctgcagt aggtgctgct cacggccacg ccgctgcact 2641 tgccgctggg gaacactctg ctgtgcaggc ttctgtcgta ggggtccagg tcggccacgc 2701 tggggctgat gatcaccagg ctctccttgg tggtcttcac ggttctcagc catctgtagt 2761 cggggtaggg gttgtgcagg ctctcctcgt atctggggtc gccggccatc ttccagttgt 2821 aggcggctct gcaggcgtcg ggggtgggtc tgaagtgctt tctcttgaag gtggtggtca 2881 cgtagcccac gaagttggtg taggtctcgg cctcggtcac cacgccggtg caggtgaagc 2941 cgttcacctt gatggccagg atgtagccca ccttcagctc catgtagctg aagccgctca 3001 ggttggtgca gccctcgtcc tccaccacca ggttgttggg gcagctcagg tggtggatgt 3061 cgatggggct ccaggggccc agcttgtcgg ggatggtgta gatggggaac ttgccgaagc 3121 acagggggaa caccagcagg ggcacgaaca gcagggcctg gggcactccg gaaggaccgg 3181 ggttactttc aacatcgcca gcgagtttca acaaagcgta gttagtacat tgaccactac 3241 cgctagcgga gaacaagtct gcctggctct tgtcaggcac cagcagctcc ttggatgcgc 3301 tttcaagact gaagatgtca gacctgcttt ttgctttgga cttcccccat gcagcgatac 3361 cacccacagc taccaggcag cacaggagca ctgcagtgat cagcatccac atgcggccgt 3421 gattcctggc tggcagagca cgtccaggag caggggcagt gggagcctct gtgccgttgt 3481 ccgtgggcac cgcggggatc gcgctggtcc cgcagcccca ctcgtcccgc ccgtcgtcgc 3541 agtcggggtg tccgtcgcac agccagtcct gcgggtaaca ctccccgtgg gcaccgggcg 3601 gctccgagca gcggaactga ccgggggggc aacgggacaa agaaccgtta ccggacccgt 3661 taccggtcac gttaccgggc agcagcagca gcagcagcgc gggcagcagc cgcgcatcga 3721 tgtcgacgca taacttcgta taggatactt tatacgaagt tatctttggg attcttccta 3781 ttttgatcca agcatcacca tcgaccctct agtccagatc tcaccatcga cccataactt 3841 cgtatagcat acattatacg aagttatgtc cctcgaagag gttcgaattc gatatcaagc 3901 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact 3961 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg 4021 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg 4081 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa 4141 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc 4201 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg 4261 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt 4321 ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt 4381 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc 4441 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc 4501 gataccgagc gctgctcgag agatctacgg gtggcatccc tgtgacccct ccccagtgcc 4561 tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag 4621 ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt 4681 atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc 4741 aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc 4801 gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag 4861 ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa 4921 ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg 4981 aaccactgct cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagcgg 5041 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact 5101 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc 5161 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg 5221 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat 5281 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 5341 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 5401 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 5461 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt 5521 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 5581 caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg 5641 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 5701 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 5761 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 5821 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 5881 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta 5941 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 6001 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 6061 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 6121 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 6181 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 6241 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 6301 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 6361 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 6421 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 6481 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 6541 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 6601 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 6661 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 6721 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 6781 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 6841 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 6901 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 6961 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 7021 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 7081 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 7141 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 7201 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 7261 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 7321 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 7381 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 7441 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 7501 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 7561 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 7621 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 7681 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 7741 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt //