LOCUS Exported 6339 bp ds-DNA linear SYN 04-JUN-2021 DEFINITION AAV-mediated expression of ultrasensitive protein calcium sensor under the CAG promoter, Cre-dependent expression. ACCESSION . VERSION . KEYWORDS pGP-AAV-CAG-FLEX-jGCaMP7c variant 1513-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6339) AUTHORS Dana H, Sun Y, Mohar B, Hulse BK, Kerlin AM, Hasseman JP, Tsegaye G, Tsang A, Wong A, Patel R, Macklin JJ, Chen Y, Konnerth A, Jayaraman V, Looger LL, Schreiter ER, Svoboda K, Kim DS TITLE High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. JOURNAL Nat Methods. 2019 Jul;16(7):649-657. doi: 10.1038/s41592-019-0435-6. Epub 2019 Jun 17. PUBMED 31209382 REFERENCE 2 (bases 1 to 6339) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 4, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6339 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 410..432 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 494..513 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind complement(548..581) /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(644..677) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 1247..1266 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1506..1527 /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1633..1654) /label=EGFP-C /note="EGFP, forward primer" CDS complement(1867..1923) /codon_start=1 /product="calmodulin-binding peptide from avian smooth muscle myosin light chain kinase" /label=calmodulin-binding peptide /translation="RRKWNKTGHAVRAIGRLSS" regulatory 1935..1944 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS complement(1951..1974) /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /label=Xpress(TM) tag /translation="DLYDDDDK" CDS complement(1978..2010) /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10" /label=T7 tag (gene 10 leader) /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS complement(2014..2031) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" regulatory 2037..2046 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 2057..2090 /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 2161..2194 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 2236..2824 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2289..2309) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2707..2718) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 2867..2988 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2878..2897) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2932..2951 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 3150..3279 /label=AAV2 ITR primer_bind complement(3345..3362) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3516..4104) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3596..3615) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4275..5135) /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 4898..4917 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5136..5240) /gene="bla" /label=AmpR promoter rep_origin complement(5266..5721) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5403..5424) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5615..5634 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 5793..5922 /label=AAV2 ITR ORIGIN 1 gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc 61 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg 121 cgcggcgggc gggagtcgct gcgcgckgcc ttcgccccgt gccccgctcc gccgccgcct 181 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg 241 gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt 301 ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg 361 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc 421 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc 481 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg 541 gatccccata acttcgtata aagtatccta tacgaagtta tatcaaaata ggaagaccaa 601 tgcttcacca tcgacccgaa ttgccaagca tcaccatcga cccataactt cgtataatgt 661 atgctatacg aagttatact agagctttca cttcgctgtc atcatttgta caaactcttc 721 gtagtttacc tgaccatccc catcgatgtc tgcttccctg atcatttcat caacctcttc 781 atctgttaac ttctctccaa ggttgttcat cacgtggcga agctctgctg cactgatgta 841 gccattgcca tccttatcaa acacaccgaa cgcttctcta atttcttctt cactgtccct 901 gtagctccct tttcttgcca tcattgtcag gaactcaggg aagtcgattg tgccgtcacc 961 gtcggcatct acttcattga tcatgtcctg cagctctgct tctgtggggt tctgccccag 1021 agaacgcatc accgtcccca gctccttggt tgttattgtc ccatccccgt ccttgtcaaa 1081 tagggagaaa gcctctttaa attctgcgat ctgctcttca gtcagttggt caggaaggtt 1141 gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc 1201 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc 1261 gtcgtccttg aagaagatgg tgcgctcctg gatgtagcct tcgggcatgg cggacttgaa 1321 gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag 1381 ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag 1441 ggtcagcttg ccgtaggtgg catcgccctc accctcgccg gacacgctga acttgtggcc 1501 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc 1561 cttgctcacc atgctccctc cggtaccgcc cttgtacagc tcgtccatgc cgagagtgat 1621 cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt 1681 cgaaagtttg gactgcacgc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc 1741 gatgggggtg ttctgctggt agtggtaggc gagctgcacg ccgccgtcct cgatgttgtg 1801 gcggatcttg aagttcgcct tgatgccgtt cttctgcttg tcggccttga tatagacgtt 1861 aggctgtgag ctcagccgac ctatagctct gactgcgtga cctgtcttat tccacttacg 1921 acgtgatgag tcgaccatgg tggcgagatc cttatcgtca tcgtcgtaca gatcccgacc 1981 catttgctgt ccaccagtca tgctagccat accatgatga tgatgatgat gagaacccat 2041 ggtggcggat cctagtataa cttcgtatag gatactttat acgaagttat cattgggatt 2101 cttcctattt tgatccaagc atcaccatcg accctctagt ccagatctca ccatcgaccc 2161 ataacttcgt atagcataca ttatacgaag ttatgtccct cgaagaggtt cgaattcgat 2221 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt 2281 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat 2341 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct 2401 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct 2461 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc 2521 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg 2581 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc 2641 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac 2701 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg 2761 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc 2821 ccgcatcgat accgtcgacc cgggcggccg cttcgagcag acatgataag atacattgat 2881 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 2941 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat 3001 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa 3061 aacctctaca aatgtggtaa aatcgataag gatcttccta gagcatggct acgtagataa 3121 gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc 3181 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 3241 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcattaatg aatcggccaa 3301 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 3361 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 3421 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 3481 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 3541 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 3601 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 3661 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 3721 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 3781 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 3841 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 3901 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca 3961 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 4021 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 4081 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 4141 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 4201 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 4261 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 4321 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 4381 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 4441 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 4501 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 4561 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 4621 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 4681 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 4741 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 4801 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 4861 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 4921 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 4981 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 5041 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 5101 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 5161 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 5221 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta 5281 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg 5341 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg 5401 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa 5461 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg 5521 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt 5581 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg 5641 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta 5701 atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc 5761 gatcggtgcg ggcctcttcg ctattacgcc agctgcgcgc tcgctcgctc actgaggccg 5821 cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag 5881 cgcgcagaga gggagtggcc aactccatca ctaggggttc cttgtagtta atgattaacc 5941 cgccatgcta cttatctacg tagccatgct ctaggaagag taccattgac gtcaataatg 6001 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 6061 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 6121 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 6181 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg 6241 agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat 6301 ttatttattt tttaattatt ttgtgcagcg atgggggcg //