LOCUS       sequence-450707-        6641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAAV vector for flippase(FLP)-dependent OCaMP (orange calcium 
            indicator) expression under the control of EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6641)
  AUTHORS   Aggarwal A, Baker HA, Durst CD, Chen I-W, de Chambrier P, Gonzales 
            JM, Marvin JS, Vandal M, Lundberg T, Sakoi K, Patel RH, Wang C-Y, 
            Visser F, Fouad Y, Sunil S, Wiens M, Terai T, Takahashi-Yamashiro K,
            Thompson RJ, Brown TA, Nasu Y, Nguyen MD, Gordon GRJ, McFarlane S, 
            Podgorski K, Holtmaat A, Campbell RE, Lohman AW
  TITLE     A sensitive orange fluorescent calcium ion indicator for imaging 
            neural activity
  JOURNAL   bioRxiv 2025.07.28.667269; doi: 
            https://doi.org/10.1101/2025.07.28.667269
REFERENCE   2  (bases 1 to 6641)
  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: "bioRxiv 
            2025.07.28.667269; doi: https://doi.org/10.1101/2025.07.28.667269"
COMMENT     Sequence Label: pAAV-EF1a-FLEX(FLP)-OCaMP-WPRE
FEATURES             Location/Qualifiers
     source          1..6641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     protein_bind    137..157
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        158..1336
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          389..1327
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1284..1304
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1336..1360
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    1390..1423
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(1986..2005)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(2701..2757)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             complement(join(2773..2787,2788..2796))
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             complement(2800..2832)
                     /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(2836..2853)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2878..2911)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(3020..3044)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3054..3642
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3107..3127)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3525..3536)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3672..3689)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3678..3885
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3893..4033
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3893..4022
                     /label=AAV2 ITR
     rep_origin      4108..4563
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4195..4214)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4405..4426
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4580..4599)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4699..4721
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4759..4777)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4845..4949
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4950..5018,5019..5810)
                     /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(5168..5187)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5981..6569
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6470..6489
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6631..6641,1..130)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tctagacaac tttgtataga aaagttgggc tccggtgccc gtcagtgggc
      181 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg
      241 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      301 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      361 tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg
      421 cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt
      481 gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta
      541 aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt
      601 gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta
      661 aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg
      721 ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc
      781 gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg
      841 ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc
      901 cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc
      961 gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc
     1021 gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga
     1081 ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac
     1141 gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt
     1201 ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt
     1261 tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc
     1321 atttcaggtg tcgtgacaag tttgtacaaa aaagcaggct gtaccggatc ctctagagtc
     1381 gactccggag aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga
     1441 ttgtagctgc tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag
     1501 gaacttcggc gcgccttatt acttcgctgt catcatttgt acaaactctt cgtagtttac
     1561 ctgaccatcc ccatcgatgt ctgctaccct gatcatttca tcaacctcct catctgttaa
     1621 cttctctcca aggtctgtca tcacgtggcg aagctctgct gcgccgatgt agccattgcc
     1681 gtccttatca aacacgcgga acgcttctct aatttcttct tcactgtctg tgtcattcat
     1741 ctttcttgcc atcatcgtca ggaactcagg gaagtcgaat gtgccgtcac cgtcggcatc
     1801 tacttcattg atcatgtcct gcagctctgc ttctgtgggg ttctgcccca gagaccggaa
     1861 caccgtcccc agctccttgg ttgttatcgt cccatccccg tccttgtcaa atagggagaa
     1921 agcctcttta aattctgcga tctgctcttc agtcagttgg tcacgcgtag cctcccagcc
     1981 catggtcttc ttctgcatta cggggccgtc gggggggaag ttggtgccgc gcagcttcac
     2041 cttgtagatg aatacgccgt cctgcaggga ggagtcctgg ttaacgtgaa taatgccgcc
     2101 gtcctcgaag ttcatcacgc gctcccacct gaagccctcg gggaaggaca gcttaaagta
     2161 gtcggggatg tcggctgggt gcttaatgta ggccttggag ccgtatgtga actgagggga
     2221 caggatgtcc caggcgaagg gcagggggcc acccttggtc accttcagct tagcggtctg
     2281 aaaggcctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt ggccgttcac
     2341 ggagccctcc atgtgcacct tgaagcgcat gaactccttg atgatggcca tgttatcctc
     2401 ctcgcccttg ctcaccagac tccctcctgt acctcccttg tacagctcgt ccctgccgcc
     2461 ggtggggtgg cggccctcgg cgcgttcgca ctgttccacg atggtgtagt cctcgttgtg
     2521 ggacacgatg tccaacttta tgtcgacgat gtaggcgccg ggcagctgca cgggcttctt
     2581 ggccttgtag gtggtcttga cctcggcggc gtagtggccg ccgtccttca gcctcagccc
     2641 cttcttgatc tcgctcttca ggacgccgtc ctcggggtac atccgctcgg aaaccacggg
     2701 tgagctcagc cgacctatag ctctgactgc gtgacctgcc ttgttccact tacgacggga
     2761 tccccatcga tccttatcgt catcgtcgta cagatcccga cccatttgct gtccaccagt
     2821 catgctagcc ataccatgat gatgatgatg atgagaaccc cgcatggtgg cgctagcgaa
     2881 gttcctatac tttctagaga ataggaactt cttgccttaa cccagaaatt atcactgtta
     2941 ttctttagaa tggtgcaaag agaagttcct ataccttttg aagaatagga acttcgaatt
     3001 cgatatcaag cttatcgata cccagctttc ttgtacaaag tgggaattcc gataatcaac
     3061 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     3121 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     3181 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     3241 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     3301 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     3361 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     3421 ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg
     3481 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     3541 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     3601 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgggaa ttcctagagc
     3661 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3721 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3781 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3841 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagggcc gcaggaaccc
     3901 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     3961 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4021 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4081 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4141 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     4201 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4261 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4321 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4381 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     4441 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     4501 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     4561 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     4621 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4681 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4741 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     4801 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     4861 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     4921 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4981 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5041 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5101 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5161 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5221 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5281 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5341 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5401 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5461 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5521 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5581 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5641 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
     5701 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     5761 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     5821 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5881 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5941 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6001 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6061 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6121 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6181 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6241 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6301 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6361 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6421 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6481 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6541 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6601 tggccttttg ctggcctttt gctcacatgt cctgcaggca g
//
