LOCUS       sequence-396539-        6950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV virus production for Cre recombinase dependent expression of 
            eGCaMP(2+).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6950)
  AUTHORS   Wait SJ, Rappleye M, Lee JD, Goy ME, Smith N, Berndt A
  TITLE     Machine Learning Ensemble Directed Engineering of Genetically 
            Encoded Fluorescent Calcium Indicators.
  JOURNAL   Res Sq. 2023 Aug 7:rs.3.rs-3146778. doi: 
            10.21203/rs.3.rs-3146778/v1. Preprint.
  PUBMED    37609342
REFERENCE   2  (bases 1 to 6950)
  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: "Res Sq. 
            2023 Aug 7:rs.3.rs-3146778. doi: 10.21203/rs.3.rs-3146778/v1. 
            Preprint."
COMMENT     Sequence Label: pAAV-ef1a-DIO-eGCaMP(2+)
FEATURES             Location/Qualifiers
     source          1..6950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      781..1369
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1270..1289
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1431..1571
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1442..1571
                     /label=AAV2 ITR
     promoter        1672..2849
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1903..2840
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2797..2817
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(2874..2907)
                     /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(2958..2991)
                     /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(3000..3443,3444..3449,3450..3884,3885..3902,
                     3903..4172,4173..4178,4179..4235,4236..4262,4263..4286,
                     4287..4289,4290..4322,4323..4325,4326..4343,4344..4352))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRVIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDDLTEEQIAEFKEHFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     3559..3578
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3818..3839
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3945..3966)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      4247..4256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4349..4358
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4365..4398
                     /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    4449..4482
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4507..5095
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4560..4580)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4978..4989)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5127..5603
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5326..5345)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5643..5783
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5643..5772
                     /label=AAV2 ITR
     rep_origin      5858..6313
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5945..5964)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6155..6176
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6330..6349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6449..6471
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6509..6527)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6595..6699
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6700..6768,6769..6950,1..610)
                     /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(6918..6937)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
       61 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
      121 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
      181 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
      241 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
      301 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
      361 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
      421 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
      481 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
      541 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
      601 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
      661 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
      721 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
      781 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
      841 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      901 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      961 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1021 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1081 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1141 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1201 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1261 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     1321 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     1381 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
     1441 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
     1501 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
     1561 taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt ttaaacagag
     1621 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagttctaga tggctccggt
     1681 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     1741 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     1801 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     1861 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
     1921 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccact
     1981 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
     2041 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
     2101 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
     2161 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
     2221 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
     2281 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
     2341 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
     2401 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     2461 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     2521 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     2581 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     2641 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     2701 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     2761 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     2821 gtttttttct tccatttcag gtgtcgtgag gtaccggatc cgtcgactcc ggaataactt
     2881 cgtataggat actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag
     2941 caatatgaaa cctcttaata acttcgtata gcatacatta tacgaagtta tggcgcgcct
     3001 tacttcgctg tcatcatttg tacaaactct tcgtagttta cctgaccatc cccatcgatg
     3061 tctgcttccc tgatcatttc atcaacctct tcatctgtta acttctctcc aaggtttgtc
     3121 atcacgtggc gaagctctgc tgcactgatg tagccattgc catccttatc aaacacaccg
     3181 aacgcttctc taatttcttc ttccgtgtcc ctgtatttca tttttcttgc catcattgtc
     3241 aggaactcag ggaagtcgat tgtgccgtca ccgtcggcat ctacttcatt gatcatgtcc
     3301 tgcagctctg cttctgtggg gttctgcccc agagaccgca tcaccgtccc cagctccttg
     3361 gttgttattg tcccatcccc gtccttgtca aatagggaga agtgctcttt aaattctgcg
     3421 atctgctctt cagtcagatc gtcaggaagg ttgtactcca gcttgtgccc caggatgttg
     3481 ccgtcctcct tgaagtcgat gcccttaagc tcgatgcggt tcaccagggt gtcgccctcg
     3541 aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc
     3601 tggatgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg
     3661 tagcggctga agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg
     3721 ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc
     3781 tcaccctcgc cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg
     3841 atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatgctccc tccggtaccg
     3901 cccttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg
     3961 accatgtgat cgcgcttctc gttggggtct ttcgaaagtt tggactgcac gctcaggtag
     4021 tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtag
     4081 gcgagctgca cgccgccgtc ctcgatgttg tggcggatgt ggaagttcgc cttgatgccg
     4141 ttcttctgct tgtcggcctt gatatagacg ttctcgagtg agctcagccg acctatcact
     4201 ctgactgcgt gacctgtctt attccactta cgacgtgatg agtcgaccat ggtggcgaga
     4261 tccttatcgt catcgtcgta cagatcccga cccatttgct gtccaccagt catgcttgcc
     4321 ataccatgat gatgatgatg atgagaaccc atggtggcgc tagcataact tcgtataaag
     4381 tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg
     4441 tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt
     4501 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     4561 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     4621 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     4681 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     4741 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     4801 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     4861 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     4921 ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     4981 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     5041 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     5101 taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
     5161 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
     5221 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
     5281 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
     5341 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
     5401 ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
     5461 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
     5521 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
     5581 ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc
     5641 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     5701 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5761 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     5821 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     5881 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     5941 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     6001 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     6061 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     6121 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     6181 acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
     6241 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     6301 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6361 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6421 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     6481 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     6541 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     6601 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6661 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6721 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6781 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6841 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6901 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
//
