LOCUS Exported 6647 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION express FLPo and GFP under the control of human synapsin promoter. ACCESSION . VERSION . KEYWORDS pAAV-hsyn-FLPo-T2A-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6647) AUTHORS An JJ, Kinney CE, Tan JW, Liao GY, Kremer EJ, Xu B TITLE TrkB-expressing paraventricular hypothalamic neurons suppress appetite through multiple neurocircuits. JOURNAL Nat Commun. 2020 Apr 7;11(1):1729. doi: 10.1038/s41467-020-15537-w. PUBMED 32265438 REFERENCE 2 (bases 1 to 6647) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6647 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 2..131 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" polyA_signal 187..235 /note="synthetic polyadenylation signal" misc_feature 249..340 /label=pause site /note="RNA polymerase II transcriptional pause signal from the human alpha-2 globin gene" primer_bind 289..308 /label=RVprimer3 /note="pGL3 vector, forward primer" promoter 376..823 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" regulatory 854..863 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 860..2155 /codon_start=1 /product="nuclear-targeted site-specific recombinase" /label=FLPo /note="FLPo is a mouse codon-optimized fusion between the SV40 nuclear localization signal and a thermostable version of the Saccharomyces cerevisiae site-specific recombinase FLP. Recombination occurs at FRT sequences." /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI" CDS 2222..2275 /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 2276..2992 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 2276..2989 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(2318..2339) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2579..2598) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2926..2947 /label=EGFP-C /note="EGFP, forward primer" misc_feature 3005..3593 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3058..3078) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3476..3487) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3626..3643) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3632..3839 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 3848..3977 /label=AAV2 ITR rep_origin 4076..4531 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4163..4182) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4373..4394 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4598..4617) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4717..4739 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4777..4795) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4863..4967 /gene="bla" /label=AmpR promoter CDS 4968..5828 /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(5186..5205) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5999..6587 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6488..6507 /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 cgcgttctag aaatattaag gtacgggagg tacttggagc 181 ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg 241 atagtactaa catacgctct ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag 301 gctgtcccca gtgcaagtgc aggtgccaga acatttctct attctagact cgagctgcag 361 agggccctgc gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc 421 ctacctgacg accgaccccg acccactgga caagcaccca acccccattc cccaaattgc 481 gcatccccta tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc 541 agcttcagca ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct 601 cagcactgaa ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca 661 ccttggtcgc gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat 721 aggggggcac gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc 781 tgcggtgggc agcggaggag tcgtgtcgtg cctgagagcg cagtcgaatt caagctgcta 841 gcaaggatcc gccgccacca tggctcctaa gaagaagagg aaggtgatga gccagttcga 901 catcctgtgc aagacccccc ccaaggtgct ggtgcggcag ttcgtggaga gattcgagag 961 gcccagcggc gagaagatcg ccagctgtgc cgccgagctg acctacctgt gctggatgat 1021 cacccacaac ggcaccgcca tcaagagggc caccttcatg agctacaaca ccatcatcag 1081 caacagcctg agcttcgaca tcgtgaacaa gagcctgcag ttcaagtaca agacccagaa 1141 ggccaccatc ctggaggcca gcctgaagaa gctgatcccc gcctgggagt tcaccatcat 1201 cccttacaac ggccagaagc accagagcga catcaccgac atcgtgtcca gcctgcagct 1261 gcagttcgag agcagcgagg aggccgacaa gggcaacagc cacagcaaga agatgctgaa 1321 ggccctgctg tccgagggcg agagcatctg ggagatcacc gagaagatcc tgaacagctt 1381 cgagtacacc agcaggttca ccaagaccaa gaccctgtac cagttcctgt tcctggccac 1441 attcatcaac tgcggcaggt tcagcgacat caagaacgtg gaccccaaga gcttcaagct 1501 ggtgcagaac aagtacctgg gcgtgatcat tcagtgcctg gtgaccgaga ccaagacaag 1561 cgtgtccagg cacatctact ttttcagcgc cagaggcagg atcgaccccc tggtgtacct 1621 ggacgagttc ctgaggaaca gcgagcccgt gctgaagaga gtgaacagga ccggcaacag 1681 cagcagcaac aagcaggagt accagctgct gaaggacaac ctggtgcgca gctacaacaa 1741 ggccctgaag aagaacgccc cctaccccat cttcgctatc aagaacggcc ctaagagcca 1801 catcggcagg cacctgatga ccagctttct gagcatgaag ggcctgaccg agctgacaaa 1861 cgtggtgggc aactggagcg acaagagggc ctccgccgtg gccaggacca cctacaccca 1921 ccagatcacc gccatccccg accactactt cgccctggtg tccaggtact acgcctacga 1981 ccccatcagc aaggagatga tcgccctgaa ggacgagacc aaccccatcg aggagtggca 2041 gcacatcgag cagctgaagg gcagcgccga gggcagcatc agataccccg cctggaacgg 2101 catcatcagc caggaggtgc tggactacct gagcagctac atcaacaggc ggatcaagct 2161 taggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaaggaat tcggcagtgg 2221 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccagtgag 2281 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 2341 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 2401 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 2461 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 2521 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 2581 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 2641 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 2701 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 2761 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 2821 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 2881 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 2941 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaaagcttat 3001 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 3061 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 3121 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga 3181 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc 3241 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct 3301 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg 3361 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct 3421 gctcgcctgt gttgccacct ggattctgcg cgggacgccc ttctgctacg tcccttcggc 3481 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg 3541 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata 3601 ccgtcgacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt 3661 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc 3721 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt 3781 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggag 3841 agatctgagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 3901 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg 3961 agcgagcgag cgcgcagaga gggccccccc ccccccccct gcagcctggc gtaatagcga 4021 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg 4081 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 4141 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 4201 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 4261 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg 4321 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 4381 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg 4441 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 4501 aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc 4561 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 4621 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 4681 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 4741 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 4801 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 4861 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 4921 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 4981 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 5041 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 5101 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 5161 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 5221 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 5281 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 5341 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 5401 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 5461 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 5521 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 5581 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 5641 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 5701 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 5761 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 5821 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 5881 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 5941 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 6001 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 6061 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 6121 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 6181 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 6241 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 6301 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 6361 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 6421 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 6481 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 6541 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 6601 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatg //