LOCUS Exported 7013 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses JRGECO1a under control of eukaryotic Ef1a promoter after Flp-dependent recombination.. ACCESSION . VERSION . KEYWORDS pAAV-Ef1a-fDIO-JRGECO1a SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7013) TITLE AAV constructs for Flp-dependent expression of fluorescent proteins and calcium indicators JOURNAL Unpublished REFERENCE 2 (bases 1 to 7013) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7013 /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 231..1409 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 462..1400 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1357..1377 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" protein_bind 1440..1473 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" primer_bind complement(2036..2055) /label=mCherry-F /note="mCherry, forward primer" CDS complement(2751..2807) /codon_start=1 /product="calmodulin-binding peptide from avian smooth muscle myosin light chain kinase" /label=calmodulin-binding peptide /translation="RRKWNKAGHAVRAIGRLSS" CDS complement(2835..2858) /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /label=Xpress(TM) tag /translation="DLYDDDDK" CDS complement(2862..2894) /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(2898..2915) /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" protein_bind complement(2998..3031) /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" misc_feature 3140..3728 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3193..3213) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3611..3622) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3760..4236 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3959..3978) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4276..4416 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4276..4405 /label=AAV2 ITR rep_origin 4491..4946 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4578..4597) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4788..4809 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4963..4982) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5082..5104 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5142..5160) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5228..5332 /gene="bla" /label=AmpR promoter CDS 5333..6193 /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(5551..5570) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6364..6952 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6853..6872 /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga 181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg 241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg 301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt 361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg 421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt 481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct 541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga 601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct 661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt 721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc 781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg 841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac 901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc 961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag 1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct 1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg 1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga 1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc 1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg 1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa 1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggag 1441 aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga ttgtagctgc 1501 tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag gaacttcggc 1561 gcggccgcct acttcgctgt catcatttgt acaaactctt cgtagtttac ctgaccatcc 1621 ccatcgatgt ctgctaccct gatcatttca tcaacctcct catctgttaa cttctctcca 1681 aggtctgtca tcacgtggcg aagctctgct gcgccgatgt agccattgcc gtccttatca 1741 aacacgcgga acgcttcgcg aatttcctct tcactgtctg tgtcattcat ttttcttgcc 1801 atcatcgtca ggaactcagg gaagtcgaat gtgccgtcac cgtcggcatc tacttcattg 1861 atcatgtcct gcagctctgc ttctgtgggg ttctgcccca gcgatcggaa caccgtccca 1921 agctccttgg ttgttatcgt cccatccccg tccttgtcaa atagggagaa agcctcttta 1981 aattctgcga tctgctcttc agtcaggtcg tcacgcgtag cctcccagcc catggtcttc 2041 ttctgcatta cggggccgtc gggggggaag ttggtgccgc gcagcttcac cttgtagatg 2101 aatacgccgt cctgcaggga ggagtcctgg ttcacgtgaa taatgccgcc gtcctcgaag 2161 ttcatcacgc gctcccacct gaagccctcg gggaaggaca gcttgaagta gtcggggatg 2221 tcggctgggt gcttaatgta ggccttggag ccgtacatga actgagggga caggatgtcc 2281 caggcgaagg gcagggggcc acccttggtc accttcagct tagcggtctg aaaggcctcg 2341 taggggcggc cctcgccctc gccctcgatc tcgaactcgt ggccgttcac ggagccctcc 2401 atgtgcacct tgaagcgcat gaactccttg atgatggcca tgttatcctc ctcgcccttg 2461 ctcaccagac tcccgcctgt acctcccttg tacagctcgt ccatgccgcc ggtggagtgg 2521 cggccctcgg cgcgttcgca ctgttccacg atggtgtagt cctcgttgtg ggacacgatg 2581 tccaacttga tgtccacgat gtaggcgccg ggcagctgca cgggcttctt ggccttgtag 2641 gtggtcttga cctcggcggc gtagtggccg ccgtccttca gcctcagccc cttcttgatc 2701 tcgctcttca gggcgccgtc ctcggggtac atccgctcgg aaaccacggg tgagctcagc 2761 cgacctatag ctctgactgc gtgacctgcc ttattccact tacgacgtga tgagtcgacc 2821 attgttgcga gatccttatc gtcatcgtcg tacagatccc gacccatttg ctgtccacca 2881 gtcatgctag ccataccatg atgatgatga tgatgagaac ctcctccagt cttgttaata 2941 tcaagtccag ccaacttaag agcaagctcg ttctgcagca tggtggcgag atctagcgaa 3001 gttcctatac tttctagaga ataggaactt cttgccttaa cccagaaatt atcactgtta 3061 ttctttagaa tggtgcaaag agaagttcct ataccttttg aagaatagga acttcgaatt 3121 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg 3181 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta 3241 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct 3301 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt 3361 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac 3421 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg 3481 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc 3541 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg 3601 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct 3661 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc 3721 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc 3781 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta 3841 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg 3901 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc 3961 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc 4021 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg 4081 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg 4141 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga 4201 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc 4261 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 4321 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 4381 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc 4441 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg 4501 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 4561 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 4621 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 4681 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg 4741 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 4801 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt 4861 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 4921 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc 4981 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 5041 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 5101 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 5161 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 5221 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 5281 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 5341 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 5401 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 5461 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 5521 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 5581 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 5641 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 5701 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 5761 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 5821 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 5881 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 5941 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 6001 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 6061 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 6121 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 6181 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 6241 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 6301 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 6361 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 6421 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 6481 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 6541 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 6601 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 6661 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 6721 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 6781 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 6841 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 6901 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 6961 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt //