LOCUS       sequence-374267-        7558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent GCaMP7f under the GFAP promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7558)
  AUTHORS   Agnew-Svoboda W, Ubina T, Figueroa Z, Wong YC, Vizcarra EA, Roebini 
            B, Wilson EH, Fiacco TA, Riccomagno MM
  TITLE     A genetic tool for the longitudinal study of a subset of 
            post-inflammatory reactive astrocytes.
  JOURNAL   Cell Rep Methods. 2022 Aug 22;2(8):100276. doi: 
            10.1016/j.crmeth.2022.100276. eCollection 2022 Aug 22.
  PUBMED    36046623
REFERENCE   2  (bases 1 to 7558)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep 
            Methods."; date: "2022-08-22"; volume: "2(8):100276. doi"; pages: " 
            10.1016/j.crmeth.2022.100276. eCollection 2022 Aug 22"
COMMENT     Sequence Label: AAV-GFAP(long)-FLEX-GCaMP7f
FEATURES             Location/Qualifiers
     source          1..7558
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     CDS             complement(166..177)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     protein_bind    2346..2370
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2371..2404)
                     /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)."
     protein_bind    complement(2437..2470)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(2483..2926,2927..2932,2933..3367,3368..3385,
                     3386..3655,3656..3661,3662..3718,3719..3745,3746..3769,
                     3770..3772,3773..3805,3806..3808,3809..3826,3827..3835))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     3042..3061
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3301..3322
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3428..3449)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      3730..3739
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      3832..3841
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3848..3881
                     /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)."
     protein_bind    3914..3947
                     /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(3948..3972)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3982..4570
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4035..4055)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4453..4464)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4600..4617)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4606..4813
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4821..4961
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4821..4950
                     /label=AAV2 ITR
     rep_origin      5036..5491
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5123..5142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5333..5354
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5508..5527)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5627..5649
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5687..5705)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5773..5877
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5878..5946,5947..6738)
                     /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"
     rep_origin      6909..7497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7398..7417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgga gctcccacct
      181 ccctctctgt gctgggactc acagagggag acctcaggag gcagtctgtc catcacatgt
      241 ccaaatgcag agcataccct gggctgggcg cagtggcgca caactgtaat tccagcactt
      301 tgggaggctg atgtggaagg atcacttgag cccagaagtt ctagaccagc ctgggcaaca
      361 tggcaagacc ctatctctac aaaaaaagtt aaaaaatcag ccacgtgtgg tgacacacac
      421 ctgtagtccc agctattcag gaggctgagg tgaggggatc acttaaggct gggaggttga
      481 ggctgcagtg agtcgtggtt gcgccactgc actccagcct gggcaacagt gagaccctgt
      541 ctcaaaagac aaaaaaaaaa aaaaaaaaaa aagaacatat cctggtgtgg agtaggggac
      601 gctgctctga cagaggctcg ggggcctgag ctggctctgt gagctgggga ggaggcagac
      661 agccaggcct tgtctgcaag cagacctggc agcattgggc tggccgcccc ccagggcctc
      721 ctcttcatgc ccagtgaatg actcaccttg gcacagacac aatgttcggg gtgggcacag
      781 tgcctgcttc ccgccgcacc ccagcccccc tcaaatgcct tccgagaagc ccattgagca
      841 gggggcttgc attgcacccc agcctgacag cctggcatct tgggataaaa gcagcacagc
      901 cccctagggg ctgcccttgc tgtgtggcgc caccggcggt ggagaacaag gctctattca
      961 gcctgtgccc aggaaagggg atcaggggat gcccaggcat ggacagtggg tggcaggggg
     1021 ggagaggagg gctgtctgct tcccagaagt ccaaggacac aaatgggtga ggggactggg
     1081 cagggttctg accctgtggg accagagtgg agggcgtaga tggacctgaa gtctccaggg
     1141 acaacagggc ccaggtctca ggctcctagt tgggcccagt ggctccagcg tttccaaacc
     1201 catccatccc cagaggttct tcccatctct ccaggctgat gtgtgggaac tcgaggaaat
     1261 aaatctccag tgggagacgg aggggtggcc agggaaacgg ggcgctgcag gaataaagac
     1321 gagccagcac agccagctca tgcgtaacgg ctttgtggag ctgtcaaggc ctggtctctg
     1381 ggagagaggc acagggaggc cagacaagga aggggtgacc tggagggaca gatccagggg
     1441 ctaaagtcct gataaggcaa gagagtgccg gccccctctt gccctatcag gacctccact
     1501 gccacataga ggccatgatt gacccttaga caaagggctg gtgtccaatc ccagccccca
     1561 gccccagaac tccagggaat gaatgggcag agagcaggaa tgtgggacat ctgtgttcaa
     1621 gggaaggact ccaggagtct gctgggaatg aggcctagta ggaaatgagg tggcccttga
     1681 gggtacagaa caggttcatt cttcgccaaa ttcccagcac cttgcaggca cttacagctg
     1741 agtgagataa tgcctgggtt atgaaatcaa aaagttggaa agcaggtcag aggtcatctg
     1801 gtacagccct tccttccctt tttttttttt tttttttttg tgagacaagg tctctctctg
     1861 ttgcccaggc tggagtggcg caaacacagc tcactgcagc ctcaacctac tgggctcaag
     1921 caatcctcca gcctcagcct cccaaagtgc tgggattaca agcatgagcc accccactca
     1981 gccctttcct tcctttttaa ttgatgcata ataattgtaa gtattcatca tggtccaacc
     2041 aaccctttct tgacccacct tcctagagag agggtcctct tgcttcagcg gtcagggccc
     2101 cagacccatg gtctggctcc aggtaccacc tgcctcatgc aggagttggc gtgcccagga
     2161 agctctgcct ctgggcacag tgacctcagt ggggtgaggg gagctctccc catagctggg
     2221 ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg gcacccgggc
     2281 atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga gcagagccag
     2341 agcaggcaag tttgtacaaa aaagcaggct ataacttcgt ataggatact ttatacgaag
     2401 ttatcctcga gtaccggtca cgcgtacgca agcttgataa cttcgtatag catacattat
     2461 acgaagttat cctagggcca ccttacttcg ctgtcatcat ttgtacaaac tcttcgtagt
     2521 ttacctgacc atccccatcg atgtctgctt ccctgatcat ttcatcaacc tcttcatctg
     2581 ttaacttctc tccaaggttt gtcatcacgt ggcgaagctc tgctgcactg atgtagccat
     2641 tgccatcctt atcaaacaca cggaacgctt ctctaatttc ttcttcactg tctgtgtatt
     2701 tcatttttct tgccatcatt gtcaggaact cagggaagtc gattgtgccg tcaccgtcgg
     2761 catctacttc attgatcatg tcctgcagct ctgcttctgt ggggttctgc cccagagacc
     2821 gcatcaccgt ccccagctcc ttggttgtta ttgtcccatc cccgtccttg tcaaataggg
     2881 agaacagctc tttaaattct gcgatctgct cttcagtcag ttggtccggc aggttgtact
     2941 ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc gatgccctta agctcgatgc
     3001 ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt
     3061 ccttgaagaa gatggtgcgc tcctggatgt agccttcggg catggcggac ttgaagaagt
     3121 cgtgctgctt catgtggtcg gggtagcggc tgaagcactg cacgccgtag gtcagggtgg
     3181 tcacgagggt gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca
     3241 gcttgccgta ggtggcatcg ccctcaccct cgccggacac gctgaacttg tggccgttta
     3301 cgtcgccgtc cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc
     3361 tcaccatgct ccctccggta ccgcccttgt acagctcgtc catgccgaga gtgatcccgg
     3421 cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttcgaaa
     3481 gtttggactg cacgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg
     3541 gggtgttctg ctggtagtgg taggcgagct gcacgccgcc gtcctcgatg ttgtggcgga
     3601 tcttgaagtt cgccttgatg ccgttcttct gcttgtcggc cttgatatag acgttctcga
     3661 gtgagctcag ccgacctata gctctgactg cgtgacctgt cttattccac ttacgacgtg
     3721 atgagtcgac catggtggcg agatccttat cgtcatcgtc gtacagatcc cgacccattt
     3781 gctgtccacc agtcatgcta gccataccat gatgatgatg atgatgagaa cccatggtgg
     3841 ctctagaata acttcgtata aagtatccta tacgaagtta taggtacctg agctcaacat
     3901 cgatagcact agtataactt cgtataatgt atgctatacg aagttatacc cagctttctt
     3961 gtacaaagtg ggaattccga taatcaacct ctggattaca aaatttgtga aagattgact
     4021 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     4081 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     4141 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4201 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     4261 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     4321 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     4381 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     4441 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     4501 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     4561 gcctccccgc atcgggaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt
     4621 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     4681 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     4741 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca
     4801 ggcatgctgg ggagggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4861 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4921 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     4981 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     5041 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     5101 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     5161 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     5221 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     5281 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5341 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     5401 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5461 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     5521 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5581 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5641 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5701 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5761 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5821 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5881 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5941 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     6001 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     6061 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     6121 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     6181 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     6241 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     6301 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6361 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6421 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6481 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6541 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc
     6601 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6661 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6721 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6781 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     7021 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     7081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
