LOCUS       sequence-271053-        6502 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used to drive axon-enriched expression of GCaMP7b in the 
            presence of Cre recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6502)
  TITLE     AAV GCaMP6 Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6502)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-Ef1a-Flex-Axon-GCaMP7b
FEATURES             Location/Qualifiers
     source          1..6502
                     /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   1..130
                     /label=AAV2 ITR
     rep_origin      216..671
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(303..322)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     513..534
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(688..707)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     807..829
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(867..885)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        953..1057
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1058..1126,1127..1918)
                     /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(1276..1295)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2089..2676
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2577..2596
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2738..2867
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2968..4146
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3199..4137
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4094..4114
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(4182..4215)
                     /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(4266..4299)
                     /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(4311..4754,4755..4760,4761..5195,5196..5213,
                     5214..5483,5484..5489,5490..5546,5547..5573,5574..5597,
                     5598..5600,5601..5633,5634..5636,5637..5654,5655..5660))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT
                     GHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYNPPDQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRSL
                     GQNPTEAELQDMINEVDADGDGTIDFPEFLTMYARKMKYRDTEEEIREAFGVFDKDGNG
                     YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     4870..4889
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5129..5150
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5256..5277)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      5558..5567
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(join(5673..5687,5688..5696))
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             complement(5700..5732)
                     /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(5736..5753)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    5887..5920
                     /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    5971..6004
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6101..6121)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
       61 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
      121 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
      181 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
      241 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
      301 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      361 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      421 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
      481 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
      541 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
      601 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
      661 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
      721 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
      781 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
      841 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
      901 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
      961 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1021 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     1081 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     1141 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     1201 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     1261 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     1321 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     1381 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     1441 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     1501 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     1561 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     1621 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     1681 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     1741 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     1801 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     1861 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     1921 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     1981 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     2041 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2101 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2161 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2221 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2281 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2341 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2401 gggctgaacg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2461 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2521 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     2581 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     2641 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     2701 cggttcctgg ccttttgctg gccttttgct cacatgtcct gcaggcagct gcgcgctcgc
     2761 tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga
     2821 gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg gccgcacgcg
     2881 taagctttgc aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc
     2941 ttctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca
     3001 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga
     3061 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     3121 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     3181 tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg
     3241 ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat
     3301 cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct
     3361 tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg
     3421 tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga
     3481 tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg
     3541 cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc
     3601 acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct
     3661 caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg
     3721 gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc
     3781 cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca
     3841 cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag
     3901 taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta
     3961 ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa
     4021 gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga
     4081 tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg
     4141 tcgtgaggta ccgtaccgga tcctctagag tcgactccgg aataacttcg tataggatac
     4201 tttatacgaa gttatgcaga atggtagctg gattgtagct gctattagca atatgaaacc
     4261 tcttaataac ttcgtatagc atacattata cgaagttatg gcgcgcccta tcacttcgct
     4321 gtcatcattt gtacaaactc ttcgtagttt acctgaccat ccccatcgat gtctgcttcc
     4381 ctgatcattt catcaacctc ttcatctgtt aacttctctc caaggtttgt catcacgtgg
     4441 cgaagctctg ctgcactgat gtagccattg ccatccttat caaacacacc gaacgcttct
     4501 ctaatttctt cttccgtgtc cctgtatttc atttttcttg cgtacattgt caggaactca
     4561 gggaagtcga ttgtgccgtc accgtcggca tctacttcat tgatcatgtc ctgcagctct
     4621 gcttctgtgg ggttctgccc cagagaccgc atcaccgtcc ccagctcctt ggttgttatt
     4681 gtcccatccc cgtccttgtc aaatagggag aaaagctctt taaattctgc gatctgctct
     4741 tcagtcagtt ggtcaggtgg gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc
     4801 ttgaagtcga tgcccttaag ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc
     4861 tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc ctggatgtag
     4921 ccttcgggca tggcggactt gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg
     4981 aagcactgca cgccgtaggt cagggtggtc acgagggtgg gccagggcac gggcagcttg
     5041 ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc ctcaccctcg
     5101 ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag gatgggcacc
     5161 accccggtga acagctcctc gcccttgctc accatgctcc ctccggtacc gcccttgtac
     5221 agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga
     5281 tcgcgcttct cgttggggtc tttcgaaagt ttggactgca cgctcaggta gtggttgtcg
     5341 ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggta ggcgagctgc
     5401 acgccgccgt cctcgatgtt gtggcggatc ttgaagttcg ccttgatgcc gttcttctgc
     5461 ttgtcggcct tgatatagac gttctcgagt gagctcagcc gacctatagc tctgactgcg
     5521 tgacctgtct tattccactt acgacgtgat gagtcgacca tggtggcgag atccttatcg
     5581 tcatcgtcgt acagatcccg acccatttgc tgtccaccag tcatgctagc cataccatga
     5641 tgatgatgat gatgagaacc ggtggcgaga tccttatcgt catcgtcgta cagatcccga
     5701 cccatttgct gtccaccagt catgctagcc ataccatgat gatgatgatg atgagaacca
     5761 atcttttggt cctcatcatt cttttcaacc tgtttggttc ttctcataca gcacagcatg
     5821 ccaccggtgg cgggtggatc cttgctagca gcttgaatta tttaaatggc cggccgtggc
     5881 gctagcataa cttcgtataa agtatcctat acgaagttat ttgccttaac ccagaaatta
     5941 tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag
     6001 ttatgaattc gatatcaagc ttatcgatat cgaattccgc tcgagataat caacctctgg
     6061 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     6121 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     6181 tctcctcctt gtataaatcc tggttagttc ttgccacggc ggaactcatc gccgcctgcc
     6241 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgtttattt
     6301 gtgaaatttg tgatgctatt gctttatttg taaccatcta gctttatttg tgaaatttgt
     6361 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     6421 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcggccgc
     6481 cacgtgcgga ccgagcggcc gc
//
