LOCUS Exported 6288 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Encodes Cre-dependent ChR2-TdTomato under control of the TRE. ACCESSION . VERSION . KEYWORDS pAAV-TRE-DIO-ChR2-TdTomato SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6288) AUTHORS Sabatini PV, Wang J, Rupp AC, Affinati AH, Flak JN, Li C, Olson DP, Myers MG Jr TITLE tTARGIT AAVs mediate the sensitive and flexible manipulation of intersectional neuronal populations in mice. JOURNAL Elife. 2021 Mar 11;10. pii: 66835. doi: 10.7554/eLife.66835. PUBMED 33704065 REFERENCE 2 (bases 1 to 6288) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6288 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(1..456) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(138..159) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 350..369 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 531..671 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 542..671 /label=AAV2 ITR protein_bind 828..1098 /label=tetracycline response element /bound_moiety="tetracycline repressor TetR" /note="contains seven copies of the tetracycline operator tetO" promoter 1131..1169 /label=minimal CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" protein_bind 1304..1337 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind 1348..1381 /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)." CDS complement(1437..2147) /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(1517..1540) /label=DsRed1-C /note="DsRed1, forward primer" primer_bind complement(1715..1734) /label=mCherry-F /note="mCherry, forward primer" primer_bind 1984..2002 /label=mCherry-R /note="mCherry, reverse primer" regulatory 2144..2153 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS complement(2163..3089) /codon_start=1 /product="H134R mutant of an N-terminal fragment of channelrhodopsin-2 from the alga Chlamydomonas reinhardtii (Nagel et al., 2005)" /label=ChR2(H134R) /note="blue light-activated cation channel" /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI RKTTKLNIGGTEIEVETLVEDEAEAGAV" regulatory 3086..3095 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind complement(3132..3165) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(3176..3209) /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)." misc_feature 3233..3821 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3286..3306) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3704..3715) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3856..3873) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3862..4069 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 4092..4221 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" rep_origin complement(4283..4871) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4363..4382) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5042..5902) /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 5665..5684 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5903..6007) /gene="bla" /label=AmpR promoter primer_bind 6075..6093 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(6131..6153) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 6253..6272 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat 61 tttttaacca atagaccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga 121 tagagttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca 181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca 241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc 301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag 361 cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca 421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg 481 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc cctgcaggca 541 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt 601 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac 661 taggggttcc tgcggccgca cgcgaaacaa ttctgcagga attcggggcc gcggaggctg 721 gatcggtccc ggtgtcttct atggaggtca aaacagcgtg gatggcgtct ccaggcgatc 781 tgacggttca ctaaacgagc tctgcttata taggtcgagt ttaccactcc ctatcagtga 841 tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc 901 gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac cactccctat 961 cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg 1021 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact 1081 ccctatcagt gatagagaaa agtgaaagtc gagctcggta cccgggtcga ggtaggcgtg 1141 tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 1201 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggcc 1261 ccgaattcga gctcggtacc cggggatcct ctagtcgacg tcgataactt cgtatagcat 1321 acattatacg aagttatccg aatcgcaata acttcgtata ggatacttta tacgaagtta 1381 tttggcgcgc caaatttaaa taacgcaacg taccttcgag ggggcccaag gaattcttac 1441 ttgtacagct cgtccatgcc gccggtggag tggcggccct cggcgcgctc gtactgttcc 1501 acgatggtgt agtcctcgtt gtgggaggtg atgtccagct tgatgttgac gttgtaggcg 1561 ccgggcagct gcacgggctt cttggccttg taggtggtct tgacctcggc gtcgtagtgg 1621 ccgccgtcct tcagcttcag cctctgcttg atctcgccct tcagggcgcc gtcctcgggg 1681 tacatccgct cggaggaggc ctcccagccc atggtcttct tctgcattac ggggccgtcg 1741 gaggggaagt tggtgccgcg cagcttcacc ttgtagatga actcgccgtc ctgcagggag 1801 gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg ctcccacttg 1861 aagccctcgg ggaaggacag cttcaagtag tcggggatgt cggcggggtg cttcacgtag 1921 gccttggagc cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg cagggggccg 1981 cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc ctcgccctcg 2041 ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgtgcacctt gaagcgcatg 2101 aactccttga tgatggccat gttgtcctcc tcgcccttgc tcaccatggt ggcgaccggt 2161 gccacggctc cggcctcggc ttcgtcttcg acgagagtct cgacctcgat ctccgttccg 2221 ccgatgttca gtttggtggt tttgcggata tctccgtgaa tcaatatgtg ctcgtggatc 2281 aggacgcgca ggtagtgtcc caacaacccc caacaatttt tactcatcag atcaataatc 2341 gtgtgaccta cggtggagcc atagacgctc aggacgccaa aaccttcggg ccccaaaatg 2401 aagagaattg ggaacatacc ccagctcacg aaaaacagcc atgccatgcc ggtcacgacc 2461 tggcggcacc gaccctttgg cacagtatga taaccctcga tatatgcttt ggcggcgtga 2521 aaaaatgtgt tcgcgccata gcacaatcca agacaaaaga agatgacttt aacatagccg 2581 gttgccatgg cgctggtagc cccccacacg atagtcccga tgtctgagac aaggagtccc 2641 atggttctcc tgctgtagtc gttgctcagg ccggtgaggt tgctcaggcg gataaggatg 2701 acaggacaag tgagcagcca ctctgcatag cgcagccact gcacccggtg tcctgtggca 2761 aggtagagca tagagggatt cttaaactca aaaaagaact cgagaatcac cttaaccatt 2821 tcaatggcgc acacatagat ctcctcccag ccgcatgtag atttccaggt ttggtaggca 2881 tagaacatca gcagcaaaat gctgaatcct gctgcaagcc actgcaggac atttgacgcg 2941 gtctgagcgc cgttcgtgcc gcgagattca atccatccgg cacagtaaca ttgatcctca 3001 gggaccagga cggacccgtt caccaccaca ggattagtaa cgaacaaaag ttcgcgtccg 3061 acggcagaca aagcgccgcc atagtccatg gtggccgcta gctagttggc gcgccaacct 3121 ggccggcctt gataacttcg tataatgtat gctatacgaa gttatccgaa tcgcaataac 3181 ttcgtataaa gtatcctata cgaagttatc gaagatcatc aagcttatcg ataatcaacc 3241 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 3301 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 3361 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 3421 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 3481 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 3541 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3601 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt 3661 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 3721 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 3781 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct 3841 cgagagatcg atctgcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 3901 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 3961 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 4021 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggac acgtgcggac 4081 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 4141 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggcggcctca gtgagcgagc 4201 gagcgcgcag ctgcctgcag gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 4261 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 4321 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 4381 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 4441 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 4501 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 4561 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 4621 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 4681 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 4741 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 4801 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 4861 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 4921 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 4981 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 5041 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 5101 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 5161 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 5221 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 5281 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 5341 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 5401 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 5461 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 5521 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 5581 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 5641 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 5701 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 5761 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 5821 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 5881 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 5941 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 6001 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga 6061 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg 6121 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg 6181 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct 6241 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccata //