LOCUS Exported 5819 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Amplified expression of GCaMP6f in combination with TET driver. ACCESSION . VERSION . KEYWORDS pAAVTREGCaMP6f SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5819) AUTHORS Sadakane O, Masamizu Y, Watakabe A, Terada S, Ohtsuka M, Takaji M, Mizukami H, Ozawa K, Kawasaki H, Matsuzaki M, Yamamori T TITLE Long-Term Two-Photon Calcium Imaging of Neuronal Populations with Subcellular Resolution in Adult Non-human Primates. JOURNAL Cell Rep. 2015 Dec 1;13(9):1989-99. doi: 10.1016/j.celrep.2015.10.050. Epub 2015 Nov 19. PUBMED 26655910 REFERENCE 2 (bases 1 to 5819) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5819 /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 173..551 /label=TRE3G promoter /note="3rd-generation Tet-responsive promoter that can be activated by binding of Tet-On(R) 3G" protein_bind 181..199 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 217..235 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 253..271 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 289..307 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 325..343 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 361..379 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 397..415 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 460..484 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 558..567 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 564..1916 /codon_start=1 /product="improved fluorescent protein-based calcium sensor (Chen et al., 2013)" /label=GCaMP6f /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" regulatory 660..669 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 950..971 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1077..1098) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1338..1357) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" misc_feature 1946..2534 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(1999..2019) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2417..2428) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 2566..3042 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(2765..2784) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 3082..3222 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3082..3211 /label=AAV2 ITR rep_origin 3297..3752 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3384..3403) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3594..3615 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3769..3788) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3888..3910 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(3948..3966) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4034..4138 /gene="bla" /label=AmpR promoter CDS 4139..4999 /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(4357..4376) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5170..5758 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5659..5678 /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 gcgtgaggcc ctttcgtctt caagaattcc tcgagtttac 181 tccctatcag tgatagagaa cgtatgaaga gtttactccc tatcagtgat agagaacgta 241 tgcagacttt actccctatc agtgatagag aacgtataag gagtttactc cctatcagtg 301 atagagaacg tatgaccagt ttactcccta tcagtgatag agaacgtatc tacagtttac 361 tccctatcag tgatagagaa cgtatatcca gtttactccc tatcagtgat agagaacgta 421 taagctttag gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca 481 gatcgcctgg agcaattcca caacactttt gtcttatacc aactttccgt accacttcct 541 accctcgtaa agtcgacgcc accatgggtt ctcatcatca tcatcatcat ggtatggcta 601 gcatgactgg tggacagcaa atgggtcggg atctgtacga cgatgacgat aaggatctcg 661 ccaccatggt cgactcatca cgtcgtaagt ggaataagac aggtcacgca gtcagagcta 721 taggtcggct gagctcactc gagaacgtct atatcaaggc cgacaagcag aagaacggca 781 tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgcctacc 841 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc 901 tgagcgtgca gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac atggtcctgc 961 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta 1021 ccggagggag catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg 1081 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggtgagggcg 1141 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc 1201 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg 1261 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac atccaggagc 1321 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg 1381 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 1441 tcctggggca caagctggag tacaacctgc cggaccaact gactgaagag cagatcgcag 1501 aatttaaaga ggaattctcc ctatttgaca aggacgggga tgggacaata acaaccaagg 1561 agctggggac ggtgatgcgg tctctggggc agaaccccac agaagcagag ctgcaggaca 1621 tgatcaatga agtagatgcc gacggtgacg gcacaatcga cttccctgag ttcctgacaa 1681 tgatggcaag aaaaatgaaa tacagggaca cggaagaaga aattagagaa gcgttcggtg 1741 tgtttgataa ggatggcaat ggctacatca gtgcagcaga gcttcgccac gtgatgacaa 1801 accttggaga gaagttaaca gatgaagagg ttgatgaaat gatcagggaa gcagacatcg 1861 atggggatgg tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg aagtgaggat 1921 ccaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt 1981 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc 2041 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg 2101 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac 2161 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc 2221 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc 2281 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa 2341 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc 2401 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc 2461 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 2521 ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg gcatccctgt 2581 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt 2641 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg 2701 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc 2761 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc 2821 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca 2881 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg 2941 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg 3001 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt gtaggtaacc 3061 acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc 3121 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc 3181 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt 3241 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc 3301 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 3361 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 3421 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 3481 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc 3541 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 3601 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg 3661 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc 3721 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc 3781 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg 3841 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat 3901 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg 3961 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt 4021 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 4081 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 4141 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 4201 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 4261 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 4321 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 4381 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 4441 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 4501 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 4561 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 4621 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 4681 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 4741 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 4801 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 4861 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 4921 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 4981 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 5041 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 5101 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 5161 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 5221 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 5281 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg 5341 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 5401 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 5461 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 5521 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 5581 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 5641 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 5701 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 5761 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt //