LOCUS       sequence-343529-        7272 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of EBFP and tTA-InteinN in Flp recombinase positive 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7272)
  AUTHORS   Chen HS, Zhang XL, Yang RR, Wang GL, Zhu XY, Xu YF, Wang DY, Zhang 
            N, Qiu S, Zhan LJ, Shen ZM, Xu XH, Long G, Xu C
  TITLE     An intein-split transactivator for intersectional neural imaging and
            optogenetic manipulation.
  JOURNAL   Nat Commun. 2022 Jun 23;13(1):3605. doi: 10.1038/s41467-022-31255-x.
  PUBMED    35739125
REFERENCE   2  (bases 1 to 7272)
  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: "2022-06-23"; volume: "13(1):3605. doi"; pages: " 
            10.1038/s41467-022-31255-x"
COMMENT     Sequence Label: pAAV-EF1a-FRT-EBFP-tTAn
FEATURES             Location/Qualifiers
     source          1..7272
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1440..1473
                     /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)"
     CDS             complement(2466..2519)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(join(2529..3239,3240..3242,3243..3245))
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     3179..3200
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(3257..3290)
                     /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)"
     misc_feature    3399..3987
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3452..3472)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3870..3881)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4019..4495
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4218..4237)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4535..4675
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4535..4664
                     /label=AAV2 ITR
     rep_origin      4750..5205
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4837..4856)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5047..5068
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5222..5241)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5341..5363
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5401..5419)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5487..5591
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5592..5660,5661..6452)
                     /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(5810..5829)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6623..7211
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7112..7131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggag
     1441 aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag gaacttcggc
     1561 gcgccttact ccttcacgta caggcacatg ccctccttca ggccgccgct gatgttcatc
     1621 tcgccggtct gggtggggaa caggtgctcc tcgctgcaga tgatctcctt gccgtcctcc
     1681 agggtgatct tgtagctctt cttcttgctc ttggggaaca cgttcagcac ctcgttgtag
     1741 ccggtgttgc tcagcaccag gtcgcccacc tggatgttgc tgatctcctt catgccctgg
     1801 ggggtctgca cctgggtctt caggtccagg cagtagccgc tgcgggtccc gccgctttcg
     1861 cactttagct gtttctccag gccacatatg attagttcca ggccgaaaag gaaggcaggt
     1921 tcggctccct gatggtcgaa cagctcaatt gcttgtctca gaagtggggg catagaatcg
     1981 gtggtaggtg tctctctttc ctcttttgct acttgatgct cctgatcctc caatacgcag
     2041 cccagtgtaa agtggcccac ggcggacaga gcgtacagtg cgttctccag ggagaagcct
     2101 tgctgacaca ggaacgcgag ctgattttcc agggtttcgt actgtttctc tgttgggcgg
     2161 gtgccgagat gcactttagc cccgtcgcga tgtgagagga gagcacagcg gaatgacttg
     2221 gcgttgttcc gcagaaagtc ttgccatgac tcgccttcca gggggcagaa gtgggtatga
     2281 tgcctgtcca gcatctcgat tgccagggca tcgagcaggg cccgcttgtt cttcacgtgc
     2341 cagtacaggg taggctgctc aactcccagc ttttgagcga gtttccttgt cgtcaggcct
     2401 tcgataccga cttcattgag taattccaga gcagagttta tgactttgct cttgtccagt
     2461 ctagatgggc caggattctc ctcgacgtca ccgcatgtta gcagacttcc tctgccctct
     2521 ccactgccct tgtacagctc gtccatgccg agagtgatcc cggcggcggt gcggaactcc
     2581 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagcacgga ctgggtgctc
     2641 aggtagtggc tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
     2701 tggtcggcga gctgcacgct gccgtcctcc acgttgtggc ggatcttgaa gttcaccttg
     2761 atgccgttct tctgcttgac ggccatgata tagatgttgt ggctgttgaa gttgtactcc
     2821 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga cgcccttcag ctcgatgcgg
     2881 ttcactaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtaggt gccgtcgtcc
     2941 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     3001 tgctgcttca tgtggtcggg gtagcgggcg aagcactgca cgccgtggct cagggtggtc
     3061 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc
     3121 ttgccgttgg tggcatcgcc ctcgccctcg cccctcacgc tgaacttgtg gccgtttacg
     3181 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     3241 accatggtgg gctagcgaag ttcctatact ttctagagaa taggaacttc ttgccttaac
     3301 ccagaaatta tcactgttat tctttagaat ggtgcaaaga gaagttccta taccttttga
     3361 agaataggaa cttcgaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     3421 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3481 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3541 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3601 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3661 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3721 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3781 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc
     3841 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3901 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     3961 ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg
     4021 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt
     4081 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt
     4141 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac
     4201 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct
     4261 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt
     4321 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg
     4381 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg
     4441 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat
     4501 tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     4561 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     4621 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     4681 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     4741 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4801 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     4861 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4921 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     4981 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5041 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     5101 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     5161 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     5221 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     5281 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     5341 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5401 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5461 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5521 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5581 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5641 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5701 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5761 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5821 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5881 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5941 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6001 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6061 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6121 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6181 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6241 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6301 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6361 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6421 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6481 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6541 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6601 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6661 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6721 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6781 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6841 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6901 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6961 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     7021 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7081 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7141 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7201 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7261 ttgctcacat gt
//
