LOCUS       sequence-474711-        6379 bp    DNA     circular SYN 05-JUN-2025
DEFINITION  AAV-mediated expression of ultrasensitive protein calcium sensor 
            under the CAG promoter, Cre mediated flip-excision switch.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6379)
  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 6379)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-019-0435-6"; journalName: "Nat Methods"; date: 
            "2019-07"; volume: "16"; issue: "7"; pages: "649-657"
COMMENT     Sequence Label: pGP-AAV-CAG-FLEX-jGCaMP7b-WPRE
FEATURES             Location/Qualifiers
     source          1..6379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    35..68
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    139..172
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     regulatory      183..192
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(189..197,198..215,216..218,219..251,252..254,255..278,
                     279..305,306..362,363..368,369..638,639..656,657..1091,
                     1092..1097,1098..1541)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNPPDQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMYARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      285..294
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     575..596
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(702..723)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(963..982)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(1552..1585)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1648..1681)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     primer_bind     complement(1716..1735)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(1797..1819)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(2098..2375)
                     /label=chicken beta-actin promoter
     repeat_region   2686..2815
                     /label=AAV2 ITR
     rep_origin      2887..3342
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2974..2993)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3184..3205
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3368..3472
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3473..3541,3542..4333)
                     /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(3691..3710)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4504..5092
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4993..5012
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5329..5458
                     /label=AAV2 ITR
     polyA_signal    5620..5741
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5657..5676)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5711..5730
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    5784..6372
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             5890..5901
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     6299..6319
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 agcttgatat cgaattcgaa cctcttcgag ggacataact tcgtataatg tatgctatac
       61 gaagttatgg gtcgatggtg agatctggac tagagggtcg atggtgatgc ttggatcaaa
      121 ataggaagaa tcccaatgat aacttcgtat aaagtatcct atacgaagtt atactaggat
      181 ccgccaccat gggttctcat catcatcatc atcatggtat ggctagcatg actggtggac
      241 agcaaatggg tcgggatctg tacgacgatg acgataagga tctcgccacc atggtcgact
      301 catcacgtcg taagtggaat aagacaggtc acgcagtcag agctataggt cggctgagct
      361 cactcgagaa cgtctatatc aaggccgaca agcagaagaa cggcatcaag gcgaacttca
      421 agatccgcca caacatcgag gacggcggcg tgcagctcgc ctaccactac cagcagaaca
      481 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc gtgcagtcca
      541 aactttcgaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
      601 ccgccgggat cactctcggc atggacgagc tgtacaaggg cggtaccgga gggagcatgg
      661 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      721 acgtaaacgg ccacaagttc agcgtgtccg gcgagggtga gggcgatgcc acctacggca
      781 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
      841 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
      901 acgacttctt caagtccgcc atgcccgaag gctacatcca ggagcgcacc atcttcttca
      961 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1021 accgcatcga gcttaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1081 tggagtacaa cccacctgac caactgactg aagagcagat cgcagaattt aaagagcttt
     1141 tctccctatt tgacaaggac ggggatggga caataacaac caaggagctg gggacggtga
     1201 tgcggtctct ggggcagaac cccacagaag cagagctgca ggacatgatc aatgaagtag
     1261 atgccgacgg tgacggcaca atcgacttcc ctgagttcct gacaatgtac gcaagaaaaa
     1321 tgaaatacag ggacacggaa gaagaaatta gagaagcgtt cggtgtgttt gataaggatg
     1381 gcaatggcta catcagtgca gcagagcttc gccacgtgat gacaaacctt ggagagaagt
     1441 taacagatga agaggttgat gaaatgatca gggaagcaga catcgatggg gatggtcagg
     1501 taaactacga agagtttgta caaatgatga cagcgaagtg aaagctctag tataacttcg
     1561 tatagcatac attatacgaa gttatgggtc gatggtgatg cttggcaatt cgggtcgatg
     1621 gtgaagcatt ggtcttccta ttttgatata acttcgtata ggatacttta tacgaagtta
     1681 tggggatcca attctttgcc aaaatgatga gacagcacaa taaccagcac gttgcccagg
     1741 agctgtagga aaaagaagaa ggcatgaaca tggttagcag aggctctaga gccgccggtc
     1801 acacgccaga agccgaaccc cgccctgccc cgtccccccc gaaggcagcc gtccccccgc
     1861 ggacagcccc gagccgctcc ccccgcacaa agggccctcc cggagcccct caaggctttc
     1921 acgcagccac agaaaagaaa caagccgtca ttaaaccaag cgctaattac agcccggagg
     1981 agaagggccg tcccgcccgc tcacctgtgg gagtaacgcg gtcagtcaga gccggggcgg
     2041 gcggcgcgag gcggcggcgg agcggggcac ggggcgaagg cagcgcgcag cgactcccgc
     2101 ccgccgcgcg cttcgctttt tatagggccg ccgccgccgc cgcctcgcca taaaaggaaa
     2161 ctttcggagc gcgccgctct gattggctgc cgccgcacct ctccgcctcg ccccgccccg
     2221 cccctcgccc cgccccgccc cgcctggcgc gcgccccccc cccccccccg cccccatcgc
     2281 tgcacaaaat aattaaaaaa taaataaata caaaattggg ggtggggagg ggggggagat
     2341 ggggagagtg aagcagaacg tggggctcac ctcgaccatg gtaatagcga tgactaatac
     2401 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
     2461 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     2521 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     2581 tattggcgtt actatgggaa catacgtcat tattgacgtc aatggtactc ttcctagagc
     2641 atggctacgt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga
     2701 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     2761 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctggc
     2821 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     2881 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2941 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3001 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3061 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3121 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3181 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3241 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3301 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     3361 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3421 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3481 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3541 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3601 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3661 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3721 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3781 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3841 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3901 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3961 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     4021 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4081 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4141 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     4201 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4261 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4321 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4381 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4441 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4501 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4561 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4621 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4681 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4741 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4801 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4861 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4921 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4981 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5041 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5101 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5161 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5221 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     5281 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct gcgcgctcgc
     5341 tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc
     5401 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttccttg
     5461 tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag gaagatcctt
     5521 atcgatttta ccacatttgt agaggtttta cttgctttaa aaaacctccc acatctcccc
     5581 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     5641 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5701 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg ctcgaagcgg
     5761 ccgcccgggt cgacggtatc gatgcgggga ggcggcccaa agggagatcc gactcgtctg
     5821 agggcgaagg cgaagacgcg gaagaggccg cagagccggc agcaggccgc gggaaggaag
     5881 gtccgctgga ttgagggccg aagggacgta gcagaaggac gtcccgcgca gaatccaggt
     5941 ggcaacacag gcgagcagcc aaggaaagga cgatgatttc cccgacaaca ccacggaatt
     6001 gtcagtgccc aacagccgag cccctgtcca gcagcgggca aggcaggcgg cgatgagttc
     6061 cgccgtggca atagggaggg ggaaagcgaa agtcccggaa aggagctgac aggtggtggc
     6121 aatgccccaa ccagtggggg ttgcgtcagc aaacacagtg cacaccacgc cacgttgcct
     6181 gacaacgggc cacaactcct cataaagaga cagcaaccag gatttataca aggaggagaa
     6241 aatgaaagcc atacgggaag caatagcatg atacaaaggc attaaagcag cgtatccaca
     6301 tagcgtaaaa ggagcaacat agttaagaat accagtcaat ctttcacaaa ttttgtaatc
     6361 cagaggttga ttatcgata
//
