LOCUS Exported 6736 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses Cre recombinase and HA tag in a Flp dependent fashion (fDIO). ACCESSION . VERSION . KEYWORDS pAAV-EF1a-fDIO-Cre 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6736 /organism="synthetic DNA construct" /mol_type="other DNA" 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 5100..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 //