LOCUS Exported 6517 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION helper virus for monosynaptic tracing; to be coinjected with pAAV-TREtight-mTagBFP2-B19G. ACCESSION . VERSION . KEYWORDS pAAV-syn-F14F15S-splitTVA-EGFP-tTA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6517) TITLE Genetic Neuroengineering Group Plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 6517) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6517 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 388..408 /label=WPRE-R /note="WPRE, reverse primer" CDS 1067..1123 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 1124..1840 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1124..1840 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1169..1190) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1430..1449) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1777..1798 /label=EGFP-C /note="EGFP, forward primer" CDS 1850..1906 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 1907..2527 /codon_start=1 /gene="synthetic gene" /product="tetracycline repressor TetR" /label=TetR /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS" regulatory 2798..2807 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter complement(2814..3261) /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" repeat_region 3289..3418 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" rep_origin complement(3480..4068) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3560..3579) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4239..5099) /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 4862..4881 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5100..5204) /gene="bla" /label=AmpR promoter primer_bind 5272..5290 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(5328..5350) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 5450..5469 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(5486..5941) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5623..5644) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5835..5854 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 6016..6156 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6027..6156 /label=AAV2 ITR polyA_signal 6158..6382 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" misc_feature 6390..461 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" CDS 6496..6507 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" ORIGIN 1 cagaaggacg tcccgcgcag aatccaggtg gcaacacagg cgagcagcca tggaaaggac 61 gtcagcttcc ccgacaacac cacggaattg tcagtgccca acagccgagc ccctgtccag 121 cagcgggcaa ggcaggcggc gatgagttcc gccgtggcaa tagggagggg gaaagcgaaa 181 gtcccggaaa ggagctgaca ggtggtggca atgccccaac cagtgggggt tgcgtcagca 241 aacacagtgc acaccacgcc acgttgcctg acaacgggcc acaactcctc ataaagagac 301 agcaaccagg atttatacaa ggaggagaaa atgaaagcca tacgggaagc aatagcatga 361 tacaaaggca ttaaagcagc gtatccacat agcgtaaaag gagcaacata gttaagaata 421 ccagtcaatc tttcacaaat tttgtaatcc agaggttgat tccggagaag ttcctattct 481 tataggagta taggaacttc tctttgcacc attctaaaga ataacagtga taatttctgg 541 gttaaggcaa gaagttccta ttctatcaga agtataggaa cttctaggtg cgcggctgct 601 gcccgcgctg ctgctgctgc tgctgcccgg taacgtgacc ggtaacgggt ccggtaacgg 661 ttctttgtcc cgttgccccc ccggtcagtt ccgctgctcg gagccgcccg gtgcccacgg 721 ggagtgttac ccgcaggact ggctgtgcga cggacacccc gactgcgacg acgggcggga 781 cgagtggggc tgcgggacca gcgcgatccc cgcggtgccc acggacaacg gcacagaggc 841 tcccactgcc cctgctcctg gacgtgctct gccagccagg aatcacggcc gcatgtggat 901 gctgatcact gcagtgctcc tgtgctgcct ggtagctgtg ggtggtatcg ctgcatgggg 961 gaagtccaaa gcaaaaagca ggtctgacat cttcagtctt gaaagcgcat ccaaggagct 1021 gctggtgcct gacaagagcc aggcagactt gttctccgga tccggagcta ctaacttcag 1081 cctgctgaag caggctggag acgtggagga gaaccctgga cccatggtga gcaagggcga 1141 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 1201 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa 1261 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac 1321 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa 1381 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa 1441 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 1501 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 1561 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt 1621 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 1681 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 1741 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 1801 cgccgccggg atcactctcg gcatggacga gctgtacaag ggaagcggag ctactaactt 1861 cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctatgt ccagactgga 1921 caagagcaaa gtcatcaact ctgccttgga gctcctgaat gaagttggca ttgagggctt 1981 gaccaccagg aagctggccc agaagctggg tgtggagcag cctaccctgt actggcatgt 2041 gaagaacaag agggctctgc ttgatgccct ggccattgag atgttggaca ggcaccacac 2101 ccacttctgc cctctggaag gggagtcctg gcaggacttc ctgaggaaca atgccaagag 2161 cttcagatgt gccttgctct cccaccggga tggtgccaaa gttcacttgg gcaccaggcc 2221 tacagagaag cagtatgaga ccctggagaa ccagctggca ttcctgtgcc aacaaggctt 2281 ctccctggaa aatgccttgt atgccctctc tgctgtgggc cacttcacct tgggctgtgt 2341 gctggaggac caggagcacc aagttgccaa ggaggagagg gagaccccca ccactgactc 2401 catgccacca ctgctgcggc aagctattga gttgtttgac caccaagggg ctgagcctgc 2461 attccttttt ggcctggaac tgatcatctg tggcctggaa aagcagctga aatgtgagtc 2521 tggctctcac gtgcccaaaa agagaaagca cgtgcctgct gatgccttgg atgattttga 2581 cctggacatg ctgcctgctg atgccctgga tgactttgat ttggacatgc tccctgctga 2641 tgcacttgat gattttgacc tggatatgct gtgagaagtt cctatactcc tataagaata 2701 ggaacttcta agaggtttca tattgctaat agcagctaca atccagctac cattctgcga 2761 agttcctata cttctgatag aataggaact tcattaacca tggtggcggc tgactgcgct 2821 ctcaggcacg acacgactcc tccgctgccc accgcagact gaggcagcgc tgagtcgccg 2881 gcgccgcagc gcagatggtc gcgcccgtgc ccccctatct cgcgcctcgc gtggtgcggt 2941 ccggctgggc cggcggcggc gcggacgcga ccaaggtggc cgggaagggg agtttgcggg 3001 ggaccggcga gtgacgtcag cgcgccttca gtgctgaggc ggcggtggcg cgcgccgcca 3061 ggcgggggcg aaggcactgt ccgcggtgct gaagctggca gtgcgcacgc gcctcgccgc 3121 atcctgtttc ccctccccct ctctgatagg ggatgcgcaa tttggggaat gggggttggg 3181 tgcttgtcca gtgggtcggg gtcggtcgtc aggtaggcac ccccaccccg cctcatcctg 3241 gtcctaaaac ccacttgcac tcatacgcag ggccctctgc agtctagcag gaacccctag 3301 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 3361 aggtcgcccg acgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcaggac 3421 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3481 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3541 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 3601 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 3661 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 3721 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 3781 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 3841 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 3901 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 3961 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 4021 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 4081 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 4141 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 4201 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 4261 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 4321 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 4381 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 4441 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 4501 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 4561 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 4621 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 4681 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 4741 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 4801 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 4861 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 4921 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 4981 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 5041 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 5101 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 5161 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 5221 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 5281 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 5341 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 5401 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag 5461 cagattgtac tgagagtgca ccataaaatt gtaaacgtta atattttgtt aaaattcgcg 5521 ttaaattttt gttaaatcag ctcatttttt aaccaataga ccgaaatcgg caaaatccct 5581 tataaatcaa aagaatagcc cgagatagag ttgagtgttg ttccagtttg gaacaagagt 5641 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat 5701 ggcccactac gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca 5761 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac 5821 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctaaggcgct ggcaagtgta 5881 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg 5941 tactatggtt gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat 6001 accgcatcag gcgcccctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg 6061 caaagcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca 6121 gagagggagt ggccaactcc atcactaggg gttcctgcca tagagcccac cgcatcccca 6181 gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac cccacccccc 6241 agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta ttaggaaagg 6301 acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca aacaacagat 6361 ggctggcaac tagaaggcac aggatccagg cggggaggcg gcccaaaggg agatccgact 6421 cgtctgaggg cgaaggcgaa gacgcggaag aggccgcaga gccggcagca ggccgcggga 6481 aggaaggtcc gctggattga gggccgaagg gacgtag //