LOCUS       sequence-396538-        6866 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV virus production for Cre recombinase dependent expression of 
            eGCaMP(plus).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6866)
  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 6866)
  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(plus)
FEATURES             Location/Qualifiers
     source          1..6866
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(97..116)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      910..1498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1399..1418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1560..1700
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1571..1700
                     /label=AAV2 ITR
     promoter        1801..2978
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2032..2969
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2926..2946
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(3003..3036)
                     /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(3087..3120)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     3688..3707
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3947..3968
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4074..4095)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(4377..4394)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    4410..4443
                     /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    4494..4527
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4552..5140
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4605..4625)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5023..5034)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5172..5648
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5371..5390)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5688..5828
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5688..5817
                     /label=AAV2 ITR
     rep_origin      5903..6358
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5990..6009)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6200..6221
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6375..6394)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6494..6516
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6554..6572)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6640..6744
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6745..6813,6814..6866,1..739)
                     /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"
ORIGIN
        1 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
       61 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      121 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
      181 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
      241 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
      301 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
      361 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
      421 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
      481 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
      541 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
      601 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
      661 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
      721 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
      781 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
      841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
      901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
      961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1021 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtc
     1561 ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg
     1621 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa
     1681 ctccatcact aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt
     1741 taaacagaga ggaatctttg cagctaatgg accttctagg tcttgaaagg agttctagat
     1801 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     1861 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     1921 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     1981 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
     2041 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
     2101 acttccactg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     2161 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     2221 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     2281 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     2341 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     2401 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     2461 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     2521 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     2581 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     2641 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     2701 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     2761 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     2821 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     2881 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     2941 tggttcaaag tttttttctt ccatttcagg tgtcgtgagg taccggatcc gtcgactccg
     3001 gaataacttc gtataggata ctttatacga agttatgcag aatggtagct ggattgtagc
     3061 tgctattagc aatatgaaac ctcttaataa cttcgtatag catacattat acgaagttat
     3121 ggcgcgcctt acttcgctgt catcatttgt acaaactctt cgtagtttac ctgaccatcc
     3181 ccatcgatgt ctgcttccct gatcatttca tcaacctctt catctgttaa cttctctcca
     3241 aggtttgtca tcacgtggcg aagctctgct gcactgatgt agccattgcc atccttatca
     3301 aacacaccga acgcttctct aatttcttct tccgtgtccc tgtatttcat ttttcttgcc
     3361 atcattgtca ggaactcagg gaagtcgatt gtgccgtcac cgtcggcatc tacttcattg
     3421 atcatgtcct gcagctctgc ttctgtgggg ttctgcccca gagaccgcat caccgtcacc
     3481 agctccttgg ttgttattgt cccatccccg tccttgtcaa atagggagaa gtgctcttta
     3541 aattctgcga tctgctcttc agtcagttgg tccggcaggt tgtactccag cttgtgcccc
     3601 aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg
     3661 tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg
     3721 gtgcgctcct ggatgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg
     3781 tggtcggggt agcggctgaa gcactgcacg ccgtaggtca gggtggtcac gagggtgggc
     3841 cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg
     3901 gcatcgccct caccctcgcc ggacacgctg aacttgtggc cgtttacgtc gccgtccagc
     3961 tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac catgctccct
     4021 ccggtaccgc ccttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac
     4081 tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tcgaaagttt ggactccacg
     4141 ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg
     4201 tagtggtagg cgagctgcac gccgccgtcc tcgatgttgt ggcggatgtg gaagttcgcc
     4261 ttgatgccgt tcttctgctt gtcggccttg atatagacgt tgatcttcag gcccatcagg
     4321 cggccgatga tcttcacggc ggtggccacc tccttgaagg tcttcttgcg acgcgtgtga
     4381 tgatggtgat gatgcatggt ggcgctagca taacttcgta taaagtatcc tatacgaagt
     4441 tatttgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa agaataactt
     4501 cgtataatgt atgctatacg aagttatgaa ttcgatatca agcttatcga taatcaacct
     4561 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     4621 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     4681 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     4741 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     4801 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     4861 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     4921 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctatgtt
     4981 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     5041 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     5101 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg agcgctgctc
     5161 gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag
     5221 ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg
     5281 actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag
     5341 ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg
     5401 cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc
     5461 ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt
     5521 ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga
     5581 tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc
     5641 ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt
     5701 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     5761 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     5821 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     5881 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5941 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     6001 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     6061 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     6121 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     6181 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     6241 tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     6301 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     6361 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     6421 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     6481 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     6541 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     6601 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6661 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6721 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     6781 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6841 gatgctgaag atcagttggg tgcacg
//
