LOCUS Exported 6234 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 6234) 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 6234) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6234 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 51..498 /label=hSyn promoter /note="human synapsin I promoter; confers neuron-specific expression (Kugler et al., 2003)" regulatory 512..521 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 518..1234 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 518..1234 /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(563..584) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(824..843) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1171..1192 /label=EGFP-C /note="EGFP, forward primer" CDS 1271..1714 /codon_start=1 /product="calcium-binding protein" /label=calmodulin /translation="MDQLTEEQIAEFKEAFSLLDKDGDGTITTKELGTGMRSLGQNPTE AELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAE LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK" CDS 1715..1756 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" misc_feature 2495..3083 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2548..2568) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(2966..2977) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3125..3142) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3131..3338 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 3367..3507 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3367..3496 /label=AAV2 ITR rep_origin 3582..4037 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3669..3688) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3879..3900 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4054..4073) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4173..4195 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4233..4251) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4319..4423 /gene="bla" /label=AmpR promoter CDS 4424..5284 /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(4642..4661) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5455..6043 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5944..5963 /label=pBR322ori-F /note="pBR322 origin, forward primer" repeat_region 6105..6234 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" ORIGIN 1 gcggccgcac gcgttgtaca gtgtctagac tgcagagggc cctgcgtatg agtgcaagtg 61 ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca 121 ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg 181 ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct 241 tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca 301 ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc 361 ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg 421 cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg 481 tcgtgcctga gagcgcagtc gagaaggatc cgccaccatg gtgagcaagg gcgaggagct 541 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 601 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 661 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg 721 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc 781 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 841 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 901 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 961 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 1021 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 1081 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct 1141 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 1201 cgggatcact ctcggcatgg acgagctgta caaggacctt ggatctggca caggcggatc 1261 cggttcaggc atggaccaac tgactgaaga gcagatcgca gaatttaaag aggctttctc 1321 cctactggac aaggacgggg atgggacaat aacaaccaag gagctgggga cggggatgcg 1381 gtctctgggg cagaacccca cagaagcaga gctgcaggac atgatcaatg aagtagatgc 1441 cgacggtgac ggcacaatcg acttccctga gttcctgaca atgatggcaa gaaaaatgaa 1501 atacacagac agtgaagaag aaattagaga agcgttccgt gtgtttgata aggatggcaa 1561 tggctacatc agtgcagcag agcttcgcca cgtgatgaca aaccttggag agaagttaac 1621 agatgaagag gttgatgaaa tgatcaggga agcagacatc gatggggatg gtcaggtaaa 1681 ctacgaagag tttgtacaaa tgatgacagc gaagggcaag ccgatcccta accctctgct 1741 gggcctggac agcactggtg gaagtggatc aggcagcggt ggatcttacg gaagccatgt 1801 ggattacgcc ggagaatcct tgtttaaggg accacgtgat tacaacccga tatcgagcac 1861 catttgtcat ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg gtattggatt 1921 tggtcccttc atcattacaa acaagcactt gtttagaaga aataatggaa cactgttggt 1981 ccaatcacta catggtgtat tcaaggtcaa gaacaccacg actttgcaac aacacctcat 2041 tgatgggagg gacatgataa ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa 2101 gctgaaattt agagagccac aaagggaaga gcgcatatgt cttgtgacaa ccaacttcca 2161 aactaagagc atgtctagca tggtgtcaga cactagttgc acattccctt catctgatgg 2221 catattctgg aagcattgga ttcaaaccaa ggatgggcag tgtggcaatc cattagtatc 2281 aactagagat gggttcattg ttggtataca ctcagcatcg aatttcacca acacaaacaa 2341 ttatttcaca agcgtgccga aaaacttcat ggaattgttg acaaatcagg aggcgcagca 2401 gtgggttagt ggttggcgat taaatgctga ctcagtattg tgggggggcc ataaagtttt 2461 catggtgtaa gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg 2521 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc 2581 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta 2641 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt 2701 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca 2761 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc 2821 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt 2881 gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg 2941 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg 3001 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat 3061 ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagctag agctcgctga 3121 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 3181 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 3241 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 3301 ggggaggatt gggaagacaa tagcaggcat gctggggagg taaccacgtg cggaccgagc 3361 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 3421 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 3481 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc 3541 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc 3601 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt 3661 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 3721 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 3781 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt 3841 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 3901 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc 3961 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 4021 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 4081 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 4141 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 4201 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 4261 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 4321 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 4381 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 4441 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 4501 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 4561 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 4621 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 4681 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 4741 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 4801 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 4861 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 4921 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 4981 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 5041 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 5101 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc 5161 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 5221 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 5281 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 5341 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 5401 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 5461 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 5521 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 5581 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 5641 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 5701 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 5761 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 5821 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 5881 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 5941 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 6001 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 6061 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg 6121 cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag 6181 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcct //