LOCUS       sequence-128134-        6772 bp    DNA     circular SYN 14-APR-2025
DEFINITION  An AAV packaging vector that expresses ER-retained low-affinity 
            GCaMP3 variant under control of the EF1a promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6772)
  AUTHORS   Henderson MJ, Baldwin HA, Werley CA, Boccardo S, Whitaker LR, Yan X,
            Holt GT, Schreiter ER, Looger LL, Cohen AE, Kim DS, Harvey BK
  TITLE     A Low Affinity GCaMP3 Variant (GCaMPer) for Imaging the Endoplasmic 
            Reticulum Calcium Store.
  JOURNAL   PLoS One. 2015 Oct 9;10(10):e0139273. doi: 
            10.1371/journal.pone.0139273. eCollection 2015.
  PUBMED    26451944
REFERENCE   2  (bases 1 to 6772)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2015-10-9"; volume: "10(10):e0139273. doi"; pages: " 
            10.1371/journal.pone.0139273. eCollection 2015"
COMMENT     Sequence Label: pOTTC810 - pAAV-EF1a-CRTsigpep-GCaMP3
            (D324G+D...
FEATURES             Location/Qualifiers
     source          1..6772
                     /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
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(1493..1501,1502..1519,1520..1522,1523..1555,1556..1558,
                     1559..1582,1583..1609,1610..1666,1667..1672,1673..1942,
                     1943..1960,1961..2395,2396..2398,2399..2401,2402..2842)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKGGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDAGGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKGGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGGGQVNYEEFVQMMTAK"
     CDS             join(1493..1501,1502..1519,1520..1522,1523..1555,1556..1558,
                     1559..1582,1583..1609,1610..1666,1667..1672,1673..1942,
                     1943..1960,1961..2395,2396..2398,2399..2401,2402..2842)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKGGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDAGGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKGGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGGGQVNYEEFVQMMTAK"
     regulatory      1589..1598
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1879..1900
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2006..2027)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2267..2286)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2899..3487
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2952..2972)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3370..3381)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3519..3995
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3718..3737)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4035..4175
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4035..4164
                     /label=AAV2 ITR
     rep_origin      4250..4705
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4337..4356)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4547..4568
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4722..4741)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4841..4863
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4901..4919)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4987..5091
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5092..5160,5161..5952)
                     /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(5310..5329)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6123..6711
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6612..6631
                     /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 tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc cgctagccac
     1441 catgctgcta tccgtgccgt tgctgctcgg cctcctcggc ctggccgtcg ccatgggttc
     1501 tcatcatcat catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga
     1561 tctgtacgac gatgacgata aggatctcgc caccatggtc gactcatcac gtcgtaagtg
     1621 gaataagaca ggtcacgcag tcagagctat aggtcggctg agctcactcg agaacgtcta
     1681 tatcaaggcc gacaagcaga agaacggcat caaggcgaac ttcaagatcc gccacaacat
     1741 cgaggacggc ggcgtgcagc tcgcctacca ctaccagcag aacaccccca tcggcgacgg
     1801 ccccgtgctg ctgcccgaca accactacct gagcgtgcag tccaaacttt cgaaagaccc
     1861 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1921 cggcatggac gagctgtaca agggcggtac cggagggagc atggtgagca agggcgagga
     1981 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     2041 gttcagcgtg tccggcgagg gtgagggcga tgccacctac ggcaagctga ccctgaagtt
     2101 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2161 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2221 cgccatgccc gaaggctaca tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2281 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2341 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaacacgcg
     2401 tgaccaactg actgaagagc agatcgcaga atttaaagag gctttctccc tatttgacaa
     2461 gggcggggat gggacaataa caaccaagga gctggggacg gtgatgcggt ctctggggca
     2521 gaaccccaca gaagcagagc tgcaggacat gatcaatgaa gtagatgccg gcggtgacgg
     2581 cacaatcgac ttccctgagt tcctgacaat gatggcaaga aaaatgaaag acacagacag
     2641 tgaagaagaa attagagaag cgttccgtgt gtttgataag ggtggcaatg gctacatcag
     2701 tgcagcagag cttcgccacg tgatgacaaa ccttggagag aagttaacag atgaagaggt
     2761 tgatgaaatg atcagggaag cagacatcga tgggggtggt caggtaaact acgaagagtt
     2821 tgtacaaatg atgacagcga agacagcaga aaaagatgag ttgtagggcg cgccgaattc
     2881 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     2941 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3001 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3061 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3121 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3181 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3241 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     3301 tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc
     3361 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3421 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3481 tccccgcatc gataccgagc gctgctcgag agatctacgg gtggcatccc tgtgacccct
     3541 ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa
     3601 taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga
     3661 ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct
     3721 attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct
     3781 gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga
     3841 ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc
     3901 tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat
     3961 tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta accacgtgcg
     4021 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4081 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     4141 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     4201 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     4261 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4321 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4381 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4441 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     4501 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4561 caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg
     4621 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4681 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     4741 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4801 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4861 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4921 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4981 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5041 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5101 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5161 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5221 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5281 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5341 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5401 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5461 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5521 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5581 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5641 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5701 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5761 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5821 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5881 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5941 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6001 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6061 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6121 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6181 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6241 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     6301 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6361 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6421 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6481 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6541 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6601 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6661 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6721 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
