LOCUS       sequence-235284-        6736 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cre recombinase and HA tag in a Flp dependent fashion 
            (fDIO).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6736)
  AUTHORS   Schneeberger M, Parolari L, Das Banerjee T, Bhave V, Wang P, Patel 
            B, Topilko T, Wu Z, Choi CHJ, Yu X, Pellegrino K, Engel EA, Cohen P,
            Renier N, Friedman JM, Nectow AR
  TITLE     Regulation of Energy Expenditure by Brainstem GABA Neurons.
  JOURNAL   Cell. 2019 Jun 24. pii: S0092-8674(19)30619-1. doi: 
            10.1016/j.cell.2019.05.048.
  PUBMED    31257028
REFERENCE   2  (bases 1 to 6736)
  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: "Cell. 2019 
            Jun 24. pii: S0092-8674(19)30619-1. doi: 
            10.1016/j.cell.2019.05.048."
COMMENT     Sequence Label: pAAV-EF1a-fDIO-Cre
FEATURES             Location/Qualifiers
     source          1..6736
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   45..174
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        275..1453
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          506..1444
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1401..1421
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1484..1517
                     /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(1613..1702)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             complement(2729..2749)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2765..2798)
                     /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    2907..3495
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2960..2980)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3378..3389)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3527..4003
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3726..3745)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4043..4183
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4043..4172
                     /label=AAV2 ITR
     rep_origin      4258..4713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4345..4364)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4555..4576
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4730..4749)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4849..4871
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4909..4927)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4995..5099
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5100..5168,5169..5960)
                     /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(5318..5337)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6131..6719
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6620..6639
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg
       61 cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag
      121 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc
      181 gcacgcgtaa gctttgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta
      241 atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga
      301 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
      361 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
      421 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
      481 caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct
      541 ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat
      601 tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg
      661 agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg
      721 aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa
      781 tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca
      841 agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc
      901 ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg
      961 gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc
     1021 gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct
     1081 tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg
     1141 tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc
     1201 cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc
     1261 gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga
     1321 gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga
     1381 gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt
     1441 tcaggtgtcg tgaggtaccg gatcctctag agtcgactcc ggagaagttc ctattctcta
     1501 gaaagtatag gaacttcgca gaatggtagc tggattgtag ctgctattag caatatgaaa
     1561 cctcttagaa gttcctattc ttcaaaaggt ataggaactt cggcgcgcct caagcgtaat
     1621 ctggaacgtc atatggatag gatcctgcat agtccgggac gtcataggga tagcccgcat
     1681 agtcaggaac atcgtatggg tagtcaccat cttcgagcag tctcaccatg gccccggttt
     1741 cagagtcaag attgcggata tagttcataa cgatgttcac atttgtccaa cctccagctt
     1801 gcataatctc tggtatggag actcctgccc gcgccatgtc tcgtgcggcc cccaccctag
     1861 cagaatgacc tgaccaggcg agatacctct ggccactgtc atccttggct ccgtatatga
     1921 gtcggtgggt tgcctcaaag atcccttcaa gggcccgtgt actcaattgg ctggtggcag
     1981 agggcgcggc gactccgttt tttctcactc gacagaacaa gtaattatta gggtcgtcag
     2041 ccacgccgga cactgagatc catctttcca ccagcttcgt tacgcccagg gacaaggcct
     2101 tttcaacgcc agcggtgctg accaatgtct ttgtgcgtcc aatatgaata agcatccggc
     2161 cgccgtccgt tcgactgatg tctttcactc tgatccgggc gatctcagct atgcgcagaa
     2221 gtgtgttata tgcaattccc agaaatgcga ggttcctgat gtcctggcac cgatcgctgt
     2281 tctccatcag ggatctgacc tggtcgaaat cagtccgctc aaaagcgaga gcctgcttag
     2341 ccctctcacc cgcatctacg ttttcctttc taattctcct cataacaagg gagactgcgt
     2401 ttgaatcgct aggccggggg agcccgctgc gtcgatgcag catattcagt tggcccaagt
     2461 gctgctggat agtcttgaca gccagccctc ttgcctggag atacagcagg taatcgcgca
     2521 cgtcctccgg ctcggcgggg aaccatttgc gattgttcag cttacaccag gctgcccagg
     2581 atcggcacac gctaagcagc attttccagg tgtgttcaga aaacgcctgt ctatccctaa
     2641 acatatccat gagatttttc ctgacctcat cgctagtggc atctactggc agcgcaggga
     2701 ggttctggtg aacagtcagg agattggaca ccttcctctt cttcttgggc atggtggcgc
     2761 tagcgaagtt cctatacttt ctagagaata ggaacttctt gccttaaccc agaaattatc
     2821 actgttattc tttagaatgg tgcaaagaga agttcctata ccttttgaag aataggaact
     2881 tcgaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     2941 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3001 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     3061 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     3121 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     3181 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3241 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     3301 aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt
     3361 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     3421 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     3481 gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt ggcatccctg
     3541 tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct
     3601 tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat
     3661 ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg
     3721 cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc
     3781 cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc
     3841 atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt
     3901 ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt
     3961 gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac
     4021 cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg
     4081 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4141 cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat
     4201 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg
     4261 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4321 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4381 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4441 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat
     4501 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4561 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag
     4621 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4681 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct
     4741 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     4801 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4861 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     4921 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4981 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5041 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5101 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5161 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5221 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5281 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5341 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5401 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5461 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5521 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5581 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5641 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     5701 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     5761 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     5821 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5881 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5941 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6001 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6061 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6121 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6181 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6241 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6301 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6361 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6421 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6481 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6541 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6601 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6661 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     6721 acgccagcaa cgcggc
//
