LOCUS Exported 6914 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of RFP and M87C448Y spastin in mammalian cells. ACCESSION . VERSION . KEYWORDS pIRES RED/SP M87C448Y SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6914) AUTHORS Solowska JM, D'Rozario M, Jean DC, Davidson MW, Marenda DR, Baas PW TITLE Pathogenic mutation of spastin has gain-of-function effects on microtubule dynamics. JOURNAL J Neurosci. 2014 Jan 29;34(5):1856-67. doi: 10.1523/JNEUROSCI.3309-13.2014. PUBMED 24478365 REFERENCE 2 (bases 1 to 6914) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6914 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..304 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 305..508 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 459..479 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 548..557 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" misc_feature 2198..2254 /label=MCS /note="multiple cloning site" misc_feature 2256..2842 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(2423..2440) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 2650..2669 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 2843..3520 /codon_start=1 /product="noncytotoxic tetrameric variant of DsRed fluorescent protein (Strack et al., 2008)" /label=DsRed-Express2 /note="mammalian codon-optimized" /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH LFQ" primer_bind complement(3023..3043) /label=DsRed1-N /note="DsRed1, reverse primer" primer_bind 3241..3260 /label=mCherry-F /note="mCherry, forward primer" primer_bind 3435..3458 /label=DsRed1-C /note="DsRed1, forward primer" polyA_signal 3642..3763 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3679..3698) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3733..3752 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(3770..4225) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3907..3928) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 4119..4138 /label=F1ori-R /note="F1 origin, reverse primer" promoter 4252..4356 /gene="bla" /label=AmpR promoter promoter 4358..4715 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(4381..4401) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 4566..4701 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4628..4647 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4750..5544 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(4804..4823) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5414..5433 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(5732..5751) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 5776..5823 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 6152..6740 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6641..6660 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgctt 541 ctcccgcgcc accatggcag ccaagaggag ctccggggcc gcgccagcac ctgcctcggc 601 ctcggccccg gcgccggtgc cgggcggcga ggccgagcgc gtccgagtct tccacaaaca 661 ggccttcgag tacatctcca ttgccctgcg catcgatgag gatgagaaag caggacagaa 721 ggagcaagct gtggaatggt ataagaaagg tattgaagaa ctggaaaaag gaatagctgt 781 tatagttaca ggacaaggtg aacagtgtga aagagctaga cgccttcaag ctaaaatgat 841 gactaatttg gttatggcca aggaccgctt acaacttcta gagaagatgc aaccagtttt 901 gccattttcc aagtcacaaa cggacgtcta taatgacagt actaacttgg catgccgcaa 961 tggacatctc cagtcagaaa gtggagctgt tccaaaaaga aaagacccct taacacacac 1021 tagtaattca ctgcctcgtt caaaaacagt tatgaaaact ggatctgcag gcctttcagg 1081 ccaccataga gcacctagtt acagtggttt atccatggtt tctggagtga aacagggatc 1141 tggtcctgct cctaccactc ataagggtac tccgaaaaca aataggacaa ataaaccttc 1201 tacccctaca actgctactc gtaagaaaaa agacttgaag aattttagga atgtggacag 1261 caaccttgct aaccttataa tgaatgaaat tgtggacaat ggaacagctg ttaaatttga 1321 tgatatagct ggtcaagact tggcaaaaca agcattgcaa gaaattgtta ttcttccttc 1381 tctgaggcct gagttgttca cagggcttag agctcctgcc agagggctgt tactctttgg 1441 tccacctggg aatgggaaga caatgctggc taaagcagta gctgcagaat cgaatgcaac 1501 cttctttaat ataagtgctg caagtttaac ttcaaaatac gtgggagaag gagagaaatt 1561 ggtgagggct ctttttgctg tggctcgaga acttcaacct tctataattt ttatagatga 1621 agttgatagc cttttgtatg aaagaagaga aggggagcac gatgctagta gacgcctaaa 1681 aactgaattt ctaatagaat ttgatggtgt acagtctgct ggagatgaca gagtacttgt 1741 aatgggtgca actaataggc cacaagagct tgatgaggct gttctcaggc gtttcatcaa 1801 acgggtatat gtgtctttac caaatgagga gacaagacta cttttgctta aaaatctgtt 1861 atgtaaacaa ggaagtccat tgacccaaaa agaactagca caacttgcta gaatgactga 1921 tggatactca ggaagtgacc taacagcttt ggcaaaagat gcagcactgg gtcctatccg 1981 agaactaaaa ccagaacagg tgaagaatat gtctgccagt gagatgagaa atattcgatt 2041 atctgacttc actgaatcct tgaaaaaaat aaaacgcagc gtcagccctc aaactttaga 2101 agcgtacata cgttggaaca aggactttgg agataccact gtttaaggaa atacctttgt 2161 aaacctgcag aacattttac ttaaaagagg aaacacaaga tctcgagctc aagcttcgaa 2221 ttctgcagtc gacggtaccg cgggcccggg atccgcccct ctccctcccc cccccctaac 2281 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc 2341 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg 2401 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg 2461 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc 2521 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa 2581 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa 2641 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta 2701 ccccattgta tgggatctga tctggggcct cggtacacat gctttacatg tgtttagtcg 2761 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca 2821 cgatgataat atggccacaa ccatggatag cactgagaac gtcatcaagc ccttcatgcg 2881 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg 2941 cgagggcaag ccctacgagg gcacccagac cgccaagctg caggtgacca agggcggccc 3001 cctgcccttc gcctgggaca tcctgtcccc ccagttccag tacggctcca aggtgtacgt 3061 gaagcacccc gccgacatcc ccgactacaa gaagctgtcc ttccccgagg gcttcaagtg 3121 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct 3181 gcaggacggc accttcatct accacgtgaa gttcatcggc gtgaacttcc cctccgacgg 3241 ccccgtaatg cagaagaaga ctctgggctg ggagccctcc accgagcgcc tgtacccccg 3301 cgacggcgtg ctgaagggcg agatccacaa ggcgctgaag ctgaagggcg gcggccacta 3361 cctggtggag ttcaagtcaa tctacatggc caagaagccc gtgaagctgc ccggctacta 3421 ctacgtggac tccaagctgg acatcacctc ccacaacgag gactacaccg tggtggagca 3481 gtacgagcgc gccgaggccc gccaccacct gttccagtag gcggccgcga ctctagatca 3541 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc 3601 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt 3661 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 3721 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc 3781 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa 3841 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt 3901 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg 3961 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt 4021 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga 4081 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg 4141 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg 4201 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc 4261 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 4321 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt 4381 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 4441 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 4501 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 4561 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 4621 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt 4681 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga 4741 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag 4801 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc 4861 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg 4921 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 4981 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg 5041 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct 5101 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg 5161 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat 5221 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc 5281 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg 5341 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc 5401 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct 5461 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat 5521 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga 5581 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct 5641 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg 5701 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag 5761 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg 5821 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc 5881 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg 5941 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact 6001 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 6061 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 6121 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 6181 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 6241 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 6301 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 6361 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 6421 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 6481 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 6541 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 6601 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 6661 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 6721 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 6781 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 6841 ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 6901 tatatggagt tccg //