LOCUS       sequence-397133-        6325 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double floxed red fluorescent protein mCherry in AAV production 
            vector expressed under the mammalian promoter (EF1a).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6325)
  AUTHORS   Lu X, Wang Y, Liu Z, Gou Y, Jaeger D, St-Pierre F
  TITLE     Widefield imaging of rapid pan-cortical voltage dynamics with an 
            indicator evolved for one-photon microscopy.
  JOURNAL   Nat Commun. 2023 Oct 12;14(1):6423. doi: 10.1038/s41467-023-41975-3.
  PUBMED    37828037
REFERENCE   2  (bases 1 to 6325)
  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: "Nat 
            Commun."; date: "2023-10-12"; volume: "14(1):6423. doi"; pages: " 
            10.1038/s41467-023-41975-3"
COMMENT     Sequence Label: pAAV-EF1a-DIO-mCherry-WPRE
FEATURES             Location/Qualifiers
     source          1..6325
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   2..131
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        232..1410
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          463..1401
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1358..1378
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1441..1474)
                     /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(1525..1558)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1584..2294)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1862..1881)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2131..2149
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      2291..2300
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2311..2344
                     /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    2395..2428
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2453..3041
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2506..2526)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2924..2935)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3073..3549
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3272..3291)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3589..3729
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3589..3718
                     /label=AAV2 ITR
     rep_origin      3804..4259
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3891..3910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4101..4122
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4276..4295)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4395..4417
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4455..4473)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4541..4645
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4646..4714,4715..5506)
                     /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(4864..4883)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5677..6265
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6166..6185
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt
       61 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      121 taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt ttaaacagag
      181 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
      241 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
      301 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
      361 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
      421 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
      481 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
      541 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
      601 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
      661 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
      721 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
      781 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
      841 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
      901 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
      961 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     1021 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     1081 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     1141 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     1201 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     1261 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     1321 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     1381 agtttttttc ttccatttca ggtgtcgtga ggtaccggat cctctagagt cgactccgga
     1441 ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg
     1501 ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg
     1561 cgcgccctat tacttgtaca tcattacttg tacagctcgt ccatgccgcc ggtggagtgg
     1621 cggccctcgg cgcgttcgta ctgttccacg atggtgtagt cctcgttgtg ggaggtgatg
     1681 tccaacttga tgttgacgtt gtaggcgccg ggcagctgca cgggcttctt ggccttgtag
     1741 gtggtcttga cctcagcgtc gtagtggccg ccgtccttca gcttcagcct ctgcttgatc
     1801 tcgcccttca gggcgccgtc ctcggggtac atccgctcgg aggaggcctc ccagcccatg
     1861 gtcttcttct gcattacggg gccgtcggag gggaagttgg tgccgcgcag cttcaccttg
     1921 tagatgaact cgccgtcctg cagggaggag tcctgggtca cggtcaccac gccgccgtcc
     1981 tcgaagttca tcacgcgctc ccacttgaag ccctcgggga aggacagctt caagtagtcg
     2041 gggatgtcgg cggggtgctt cacgtaggcc ttggagccgt acatgaactg aggggacagg
     2101 atgtcccagg cgaagggcag ggggccaccc ttggtcacct tcagcttggc ggtctgggtg
     2161 ccctcgtagg ggcggccctc gccctcgccc tcgatctcga actcgtggcc gttcacggag
     2221 ccctccatgt gcaccttgaa gcgcatgaac tccttgatga tggccatgtt atcctcctcg
     2281 cccttgctca ccatggtggc ggtagctagc ataacttcgt ataaagtatc ctatacgaag
     2341 ttatttgcct taacccagaa attatcactg ttattcttta gaatggtgca aagaataact
     2401 tcgtataatg tatgctatac gaagttatga attcgatatc aagcttatcg ataatcaacc
     2461 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     2521 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     2581 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     2641 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     2701 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     2761 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     2821 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt
     2881 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     2941 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3001 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct
     3061 cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa
     3121 gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct
     3181 gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa
     3241 gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg
     3301 gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag
     3361 cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt
     3421 tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg
     3481 atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc
     3541 cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag
     3601 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     3661 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     3721 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3781 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3841 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     3901 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3961 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     4021 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4081 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     4141 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     4201 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     4261 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     4321 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4381 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     4441 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     4501 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     4561 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     4621 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     4681 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     4741 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     4801 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     4861 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     4921 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     4981 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5041 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5101 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     5161 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     5221 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     5281 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     5341 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     5401 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     5461 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     5521 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5581 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     5641 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5701 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5761 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5821 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5881 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5941 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6001 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6061 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6121 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6181 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6241 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6301 cttttgctgg ccttttgctc acatg
//
