LOCUS       Exported                7305 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  In the presence of Cre, the plasmid can be used to express GCaMP6s 
            fused to the presynaptic protein synaptophysin. Drives enriched 
            expression of GCaMP6s in neuronal axon boutons..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-Ef1a-DIO-Synaptophysin-GCaMP6s
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7305)
  TITLE     AAV GCaMP6 Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7305)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7305
                     /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    1445..1478
                     /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    1529..1562
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1574..2926)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     2133..2152
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2392..2413
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2519..2540)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2821..2830
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             complement(2937..2960)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     protein_bind    complement(3953..3986)
                     /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(4037..4070)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(4167..4187)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4569..4709
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4569..4698
                     /label=AAV2 ITR
     rep_origin      4784..5239
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4871..4890)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5081..5102
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5256..5275)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5375..5397
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5435..5453)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5521..5625
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5626..6486
                     /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(5844..5863)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6657..7244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7145..7164
                     /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 gtaccgtacc ggatcctcta gagtcgactc
     1441 cggaataact tcgtatagga tactttatac gaagttatgc agaatggtag ctggattgta
     1501 gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt atacgaagtt
     1561 atggcgcgcc ttatcacttc gctgtcatca tttgtacaaa ctcttcgtag tttacctgac
     1621 catccccatc gatgtctgct tccctgatca tttcatcaac ctcttcatct gttaacttct
     1681 ctccaaggtt tgtcatcacg tggcgaagct ctgctgcact gatgtagcca ttgccatcct
     1741 tatcaaacac accgaacgct tctctaattt cttcttccgt gtccctgtat ttcatttttc
     1801 ttgccatcat tgtcaggaac tcagggaagt cgattgtgcc gtcaccgtcg gcatctactt
     1861 cattgatcat gtcctgcagc tctgcttctg tggggttctg ccccagagac cgcatcaccg
     1921 tccccagctc cttggttgtt attgtcccat ccccgtcctt gtcaaatagg gagaaagcct
     1981 ctttaaattc tgcgatctgc tcttcagtca gttggtccgg caggttgtac tccagcttgt
     2041 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     2101 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     2161 agatggtgcg ctcctggatg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     2221 tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg
     2281 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     2341 aggtggcatc gccctcaccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     2401 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgc
     2461 tccctccggt accgcccttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     2521 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttcgaa agtttggact
     2581 gcacgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     2641 gctggtagtg gtaggcgagc tgcacgccgc cgtcctcgat gttgtggcgg atgtggaagt
     2701 tcgccttgat gccgttcttc tgcttgtcgg ccttgatata gacgttctcg agtgagctca
     2761 gccgacctat agctctgact gcgtgacctg tcttattcca cttacgacgt gatgagtcga
     2821 ccatggtggc gagatcctta tcgtcatcgt cgtacagatc ccgacccatt tgctgtccac
     2881 cagtcatgct agccatacca tgatgatgat gatgatgaga acccattggc gagatcctta
     2941 tcgtcatcgt cgtacagatc ccgacccatt tgctgtccac cagtcccggt ggatcctgat
     3001 tggagaagga ggtgggcgca ccctgttggc catagccttg ctgcccatag tcgccctgag
     3061 gcccgtagcc accgccaccg ctggctggct gcccgtaatc gggttgataa ccaccctgag
     3121 gcccgtagga gtcctggggc ccatagcctc cggggccctg cccgtagccc gcatcgccgt
     3181 aggcatcgcc aggtgctggt tgcttttccg gggcgcctgg aggtgcgcgc atgaatgggg
     3241 ctgcccagcc tgtctccttg aacacgaacc ataagttgcc aacccagagc accaggttca
     3301 ggaagccaaa caccactgtg gtgttgagtc ctgaagtcac agggtccctc agttccttgc
     3361 atgtgttccc tgtctggcgg cacatgggca tctccttgat aatgttctct gggtccgtgg
     3421 ccatcttcac atcggacagg cctttggccc aggctgatga actaactagc cacatgaaag
     3481 cgaacacggc tgtagccaga aagtccatca taggcccttt gttgttctct cggtacttgt
     3541 tctgcaggaa gatgtaggtg gccagggccc ccatggagta gaggaaggca aacacagcca
     3601 cggtgacaaa gaattcagcc gacgaggagt agtccccaac caggaagatc ttggtagtgc
     3661 cccctttgac gcaggagggt gcatcaaagt acacttggtg cagcctgaag gggtactcga
     3721 attcaacttc gatgttgagg gcactctccg tcttgttggc acactccacg ctcagccgaa
     3781 gctccccggt gtagctgcca cacgtagcaa aggcgaagat ggcaaagacc cactgcagca
     3841 ccttcacgaa gccaaggggc tccttgacca cccggaactg acccccagcc accagctgat
     3901 tcaccacgtc catgccacca agcttattta aatggccggc cgtggcgcta gcataacttc
     3961 gtataaagta tcctatacga agttatttgc cttaacccag aaattatcac tgttattctt
     4021 tagaatggtg caaagaataa cttcgtataa tgtatgctat acgaagttat gaattcgata
     4081 tcaagcttat cgatatcgaa ttccgctcga gataatcaac ctctggatta caaaatttgt
     4141 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     4201 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     4261 aaatcctggt tagttcttgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     4321 acaggggctc ggctgttggg cactgacaat tccgtggtgt ttatttgtga aatttgtgat
     4381 gctattgctt tatttgtaac catctagctt tatttgtgaa atttgtgatg ctattgcttt
     4441 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     4501 gtttcaggtt cagggggaga tgtgggaggt tttttaaagc ggccgccacg tgcggaccga
     4561 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4621 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4681 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     4741 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     4801 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4861 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4921 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4981 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5041 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5101 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5161 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5221 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5281 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5341 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5401 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     5461 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5521 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5581 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5641 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5701 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5761 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5821 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5881 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5941 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6001 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6061 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6121 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6181 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6241 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6301 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6361 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6421 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6481 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6541 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6601 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6661 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6721 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6781 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6841 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6901 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6961 cagcggtcgg gctgaacggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7021 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7081 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7141 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7201 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7261 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
