LOCUS       sequence-492083-        8430 bp    DNA     circular SYN 06-NOV-2025
DEFINITION  Flp-dependent expression of Kir2.1-T2A-tdTomato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8430)
  AUTHORS   Xue M, Atallah BV, Scanziani M
  TITLE     Equalizing excitation-inhibition ratios across visual cortical 
            neurons.
  JOURNAL   Nature. 2014 Jul 31;511(7511):596-600. doi: 10.1038/nature13321. 
            Epub 2014 Jun 22.
  PUBMED    25043046
REFERENCE   2  (bases 1 to 8430)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nature13321"; journalName: "Nature"; date: "2014-07-31- 
            31"; volume: "511"; issue: "7511"; pages: "596-600"
COMMENT     Sequence Label: pAAV-EF1-alpha-F-FLEX- Kir2.1-T2A-tdTomato
FEATURES             Location/Qualifiers
     source          1..8430
                     /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    1500..1533
                     /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(1540..2970)
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     regulatory      2967..2976
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2986..3039)
                     /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(4330..4359)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      4359..4368
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(4497..4530)
                     /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    4557..5145
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4610..4630)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5028..5039)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5177..5653
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5376..5395)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5693..5833
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5693..5822
                     /label=AAV2 ITR
     rep_origin      5908..6363
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5995..6014)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6205..6226
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6380..6399)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6499..6521
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6559..6577)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6645..6749
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6750..6818,6819..7610)
                     /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(6968..6987)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7781..8369
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8270..8289
                     /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 gtaccgaagt tcctattcta tcagaagtat
     1441 aggaacttcg cagaatggta gctggattgt agctgctatt agcaatatga aacctcttag
     1501 aagttcctat tctctagaaa gtataggaac ttcgaattct tacttgtaca gctcgtccat
     1561 gccgtacagg aacaggtggt ggcggccctc ggagcgctcg tactgttcca cgatggtgta
     1621 gtcctcgttg tgggaggtga tgtccagctt ggtgtccacg tagtagtagc cgggcagttg
     1681 cacgggcttc ttggccatgt agatggtctt gaactccacc aggtagtggc cgccgtcctt
     1741 cagcttcagg gcctggtgga tctcgccctt cagcacgccg tcgcgggggt acaggcgctc
     1801 ggtggaggcc tcccagccca tggtcttctt ctgcattacg gggccgtcgg gggggaagtt
     1861 ggtgccgcgc atcttcacct tgtagatcag cgtgccgtcc tgcagggagg agtcctgggt
     1921 cacggtcacc agaccgccgt cctcgaagtt catcacgcgc tcccacttga agccctcggg
     1981 gaaggacagc ttcttgtaat cggggatgtc ggcggggtgc ttcacgtacg ccttggagcc
     2041 gtacatgaac tggggggaca ggatgtccca ggcgaagggc agggggccgc ccttggtcac
     2101 cttcagcttg gcggtctggg tgccctcgta ggggcggccc tcgccctcgc cctcgatctc
     2161 gaactcgtgg ccgttcatgg agccctccat gcgcaccttg aagcgcatga actctttgat
     2221 gacggccatg ttgttgtcct cggaggaggc ggtgccggag ctgccgctgc cggtgctgcc
     2281 ggtgccatgc cccaggaaca ggtggtggcg gccctcggag cgctcgtact gttccacgat
     2341 ggtgtagtcc tcgttgtggg aggtgatgtc cagcttggtg tccacgtagt agtagccggg
     2401 cagttgcacg ggcttcttgg ccatgtagat ggtcttgaac tccaccaggt agtggccgcc
     2461 gtccttcagc ttcagggcct ggtggatctc gcccttcagc acgccgtcgc gggggtacag
     2521 gcgctcggtg gaggcctccc agcccatggt cttcttctgc attacggggc cgtcgggggg
     2581 gaagttggtg ccgcgcatct tcaccttgta gatcagcgtg ccgtcctgca gggaggagtc
     2641 ctgggtcacg gtcaccagac cgccgtcctc gaagttcatc acgcgctccc acttgaagcc
     2701 ctcggggaag gacagcttct tgtaatcggg gatgtcggcg gggtgcttca cgtacgcctt
     2761 ggagccgtac atgaactggg gggacaggat gtcccaggcg aagggcaggg ggccgccctt
     2821 ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc
     2881 gatctcgaac tcgtggccgt tcatggagcc ctccatgcgc accttgaagc gcatgaactc
     2941 tttgatgacc tcctcgccct tgctcaccat ggtggcggga tccgctgggc caggattctc
     3001 ctcgacgtca ccgcatgtta gcagacttcc tctgccctct ccactgccta tctccgattc
     3061 tcgccttaag ggcctgggct ctagaggtac gcttgcctgg ttgtggagat ctatgccagg
     3121 aggtgagtct gtgcttgtgc tctctgggac tccgttctca ctatcctcct cttcctcttt
     3181 gcttgttagg gcaacttcat tttcatagca aaatgaattt gcatttgaaa ggatgtattt
     3241 cttctctgct aagtctctgg cactacaaag gggggtgtta ggtacttcat aagtcttatg
     3301 gaatcttgaa tagtctactt tatagtagtg tttctcttca aagagcactg gctcatagcg
     3361 gtgaccccag agaatttcat tggccagata cgaactccgg cattgagttg tcatggcagt
     3421 cgcctccacc atgccttcca gtatgacaac aatttcaaag tctgcattgt caatgtcctg
     3481 cttactcaag tcatataaag ggctgtcttc atctatttcg tgaacgatag tgatggggga
     3541 cactagaaat atgcggtcaa ttccactatc aaaaccaaca ttgatgtcta tctggtccaa
     3601 agggataaac tccccttctg aagtgatcct agatttgaga agctgtgccc ggacatgagc
     3661 tcccacaagg tggctctttc gaaggttacc cactctccac atcaagcaga gtttgccatc
     3721 cctcatggca atcacagcat tgtgactgaa gacaagagtc tcatttctct tctttggctt
     3781 tgccatcttc gccatgactg caccaatgat gaaggcgtca atgatgcagc ctacaattga
     3841 ctggaatacc accatgaaga cagcaattgg gcattcgtct gtcacacacc tgaaaccata
     3901 gccaatggtt gtctgggtct cgatggagaa gaggaaggca gccgtgaagc tgttaacctc
     3961 cgacacgcat gctttgctca ctttagaagt atctagatcc ccatggagca gggctatcaa
     4021 ccaaaacaca cagccaaaga acagccagga gagcacgaag gcaagacaga agataaccag
     4081 catccacctc cagcggatgt cgacacaggt agtaaagatg tctgccaggt acctctgtcc
     4141 cttctcaccc acgttgataa actgaacatt gcaatgacca tctttcttca caaagcggct
     4201 cctgcactgt tgtcgggtat ggactttact cttgccattc ccaaagccat tggcaactgc
     4261 catagtggcc agcttcatgc catcttcctc cgaagagaca atgctgtacc ggttggttct
     4321 cacactgccc aggtcctcct cgctgatcag cttctgctcc atggtggcgc ggccgcggat
     4381 cccggctagc tacgtaggcg cgccttaatt aagaagttcc tatacttctg atagaatagg
     4441 aacttcttgc cttaacccag aaattatcac tgttattctt tagaatggtg caaagagaag
     4501 ttcctatact ttctagagaa taggaacttc cagaattcga tatcaagctt atcgataatc
     4561 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     4621 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     4681 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     4741 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     4801 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     4861 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     4921 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     4981 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     5041 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     5101 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
     5161 tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
     5221 ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
     5281 gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg
     5341 gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc
     5401 agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc
     5461 tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
     5521 tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca
     5581 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
     5641 cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc
     5701 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     5761 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     5821 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5881 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     5941 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     6001 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     6061 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     6121 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     6181 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     6241 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     6301 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     6361 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     6421 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     6481 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     6541 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     6601 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     6661 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     6721 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     6781 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     6841 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     6901 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6961 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     7021 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     7081 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     7141 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     7201 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     7261 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     7321 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     7381 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     7441 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     7501 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     7561 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     7621 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     7681 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     7741 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     7801 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     7861 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     7921 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7981 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     8041 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     8101 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     8161 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     8221 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     8281 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     8341 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     8401 tggccttttg ctggcctttt gctcacatgt
//
