LOCUS Exported 6233 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Neuronal expression of uFLARE protease component. ACCESSION . VERSION . KEYWORDS Syn:eGFP-CaM-uTEV1-Delta(220-242) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6233) AUTHORS Sanchez MI, Ting AY TITLE Directed evolution improves the catalytic efficiency of TEV protease. JOURNAL Nat Methods. 2019 Dec 9. pii: 10.1038/s41592-019-0665-7. doi: 10.1038/s41592-019-0665-7. PUBMED 31819267 REFERENCE 2 (bases 1 to 6233) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6233 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" promoter 181..628 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" regulatory 642..651 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 648..1364 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 648..1364 /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(693..714) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(954..973) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1301..1322 /label=EGFP-C /note="EGFP, forward primer" CDS 1401..1844 /codon_start=1 /product="calcium-binding protein" /label=calmodulin /translation="MDQLTEEQIAEFKEAFSLLDKDGDGTITTKELGTGMRSLGQNPTE AELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAE LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" CDS 1845..1886 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" misc_feature 2625..3213 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2678..2698) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3096..3107) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3255..3272) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3261..3468 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 3497..3637 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3497..3626 /label=AAV2 ITR rep_origin 3711..4166 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3798..3817) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4008..4029 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4183..4202) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4302..4324 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4362..4380) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4448..4552 /gene="bla" /label=AmpR promoter CDS 4553..5413 /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(4771..4790) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5584..6172 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6073..6092 /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 gcgttgtaca gtgtctagac tgcagagggc cctgcgtatg 181 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga 241 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga 301 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg 361 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc 421 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg 481 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg 541 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg 601 aggagtcgtg tcgtgcctga gagcgcagtc gagaaggatc cgccaccatg gtgagcaagg 661 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 721 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc 781 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 841 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct 901 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg 961 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 1021 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 1081 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga 1141 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 1201 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc 1261 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 1321 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caaggacctt ggatctggca 1381 caggcggatc cggttcaggc atggaccaac tgactgaaga gcagatcgca gaatttaaag 1441 aggctttctc cctactggac aaggacgggg atgggacaat aacaaccaag gagctgggga 1501 cggggatgcg gtctctgggg cagaacccca cagaagcaga gctgcaggac atgatcaatg 1561 aagtagatgc cgacggtgac ggcacaatcg acttccctga gttcctgaca atgatggcaa 1621 gaaaaatgaa atacacagac agtgaagaag aaattagaga agcgttccgt gtgtttgata 1681 aggatggcaa tggctacatc agtgcagcag agcttcgcca cgtgatgaca aaccttggag 1741 agaagttaac agatgaagag gttgatgaaa tgatcaggga agcagacatc gatggggatg 1801 gtcaggtaaa ctacgaagag tttgtacaaa tgatgacagc gaagggcaag ccgatcccta 1861 accctctgct gggcctggac agcactggtg gaagtggatc aggcagcggt ggatcttacg 1921 gaagccatgt ggattacgcc ggagaatcct tgtttaaggg accacgtgat tacaacccga 1981 tatcgagcac catttgtcat ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg 2041 gtattggatt tggtcccttc atcattacaa acaagcactt gtttagaaga aataatggaa 2101 cactgttggt ccaatcacta catggtgtat tcaaggtcaa gaacaccacg actttgcaac 2161 aacacctcat tgatgggagg gacatgataa ttattcgcat gcctaaggat ttcccaccat 2221 ttcctcaaaa gctgaaattt agagagccac aaagggaaga gcgcatatgt cttgtgacaa 2281 ccaacttcca aactaagagc atgtctagca tggtgtcaga cactagttgc acattccctt 2341 catctgatgg catattctgg aagcattgga ttcaaaccaa ggatgggcag tgtggcaatc 2401 cattagtatc aactagagat gggttcattg ttggtataca ctcagcatcg aatttcacca 2461 acacaaacaa ttatttcaca agcgtgccga aaaacttcat ggaattgttg acaaatcagg 2521 aggcgcagca gtgggttagt ggttggcgat taaatgctga ctcagtattg tgggggggcc 2581 ataaagtttt catggtgtaa gaattcgata tcaagcttat cgataatcaa cctctggatt 2641 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 2701 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 2761 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 2821 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 2881 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 2941 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 3001 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct 3061 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 3121 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 3181 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagctag 3241 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 3301 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 3361 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 3421 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggagg taaccacgtg 3481 cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct 3541 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg 3601 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc 3661 cttacgcatc tgtgcggtat tcacaccgca tacgtcaaag caaccatagt acgcgccctg 3721 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 3781 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 3841 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 3901 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg 3961 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 4021 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt 4081 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 4141 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc 4201 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 4261 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 4321 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 4381 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 4441 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 4501 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 4561 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 4621 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 4681 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 4741 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 4801 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 4861 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 4921 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 4981 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 5041 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 5101 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 5161 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 5221 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat 5281 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 5341 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 5401 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 5461 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 5521 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 5581 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 5641 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 5701 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 5761 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 5821 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 5881 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 5941 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 6001 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 6061 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 6121 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 6181 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt //