LOCUS Exported 6400 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses E112K spastin M1 and M87 in mammalian cells. ACCESSION . VERSION . KEYWORDS pCMV-Tag/E112K with 5'UTR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6400) AUTHORS Solowska JM, Garbern JY, Baas PW TITLE Evaluation of loss of function as an explanation for SPG4-based hereditary spastic paraplegia. JOURNAL Hum Mol Genet. 2010 Jul 15;19(14):2767-79. doi: 10.1093/hmg/ddq177. Epub 2010 Apr 29. PUBMED 20430936 REFERENCE 2 (bases 1 to 6400) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6400 /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" promoter 554..572 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(2763..2779) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2764..2780) /label=pBluescriptKS /note="For pBluescript vector" primer_bind complement(2835..2854) /label=T7 /note="T7 promoter, forward primer" promoter complement(2836..2854) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" polyA_signal 3128..3249 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3165..3184) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3219..3238 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(3256..3711) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3393..3414) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3605..3624 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3738..3842 /gene="bla" /label=AmpR promoter promoter 3844..4201 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3867..3887) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 4052..4187 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4114..4133 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4236..5030 /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(4290..4309) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4900..4919 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(5218..5237) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 5262..5309 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5638..6226 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6127..6146 /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 gctagcgatt 541 acgccaagct cgaaattaac cctcactaaa gggaacaaaa gctggaggac ccacgcgtcc 601 gggcccgagc caccgactgc aggaggagaa ggggttgtgc tcctggccga ggaaggagaa 661 aggggcgggg ccggcgggca gcgtgcggca gtgcggagct cctgagaccg gcgggcacac 721 gggggtctgt ggcccccgcc gtagcagtgg ctgccgccgt cgcttggttc ccgtcggtct 781 gcgggaggcg ggttatggcg gcggcggcag tgagagctgt gaatgaattc tccgggtgga 841 cgagggaaga agaaaggctc cggcggcgcc agcaacccgg tgcctcccag gcctccgccc 901 ccttgcctgg cccccgcccc tcccgccgcc gggccggccc ctccgcccga gtcgccgcat 961 aagcggaacc tgtactattt ctcctacccg ctgtttgtag gcttcgcgct gctgcgtttg 1021 gtcgccttcc acctggggct cctcttcgtg tggctctgcc agcgcttctc ccgcgccctc 1081 atggcagcca agaggagctc cggggccgcg ccagcacctg cctcggcctc ggccccggcg 1141 ccggtgccgg gcggcaaggc cgagcgcgtc cgagtcttcc acaaacaggc cttcgagtac 1201 atctccattg ccctgcgcat cgatgaggat gagaaagcag gacagaagga gcaagctgtg 1261 gaatggtata agaaaggtat tgaagaactg gaaaaaggaa tagctgttat agttacagga 1321 caaggtgaac agtgtgaaag agctagacgc cttcaagcta aaatgatgac taatttggtt 1381 atggccaagg accgcttaca acttctagag aagatgcaac cagttttgcc attttccaag 1441 tcacaaacgg acgtctataa tgacagtact aacttggcat gccgcaatgg acatctccag 1501 tcagaaagtg gagctgttcc aaaaagaaaa gaccccttaa cacacactag taattcactg 1561 cctcgttcaa aaacagttat gaaaactgga tctgcaggcc tttcaggcca ccatagagca 1621 cctagttaca gtggtttatc catggtttct ggagtgaaac agggatctgg tcctgctcct 1681 accactcata agggtactcc gaaaacaaat aggacaaata aaccttctac ccctacaact 1741 gctactcgta agaaaaaaga cttgaagaat tttaggaatg tggacagcaa ccttgctaac 1801 cttataatga atgaaattgt ggacaatgga acagctgtta aatttgatga tatagctggt 1861 caagacttgg caaaacaagc attgcaagaa attgttattc ttccttctct gaggcctgag 1921 ttgttcacag ggcttagagc tcctgccaga gggctgttac tctttggtcc acctgggaat 1981 gggaagacaa tgctggctaa agcagtagct gcagaatcga atgcaacctt ctttaatata 2041 agtgctgcaa gtttaacttc aaaatacgtg ggagaaggag agaaattggt gagggctctt 2101 tttgctgtgg ctcgagaact tcaaccttct ataattttta tagatgaagt tgatagcctt 2161 ttgtgtgaaa gaagagaagg ggagcacgat gctagtagac gcctaaaaac tgaatttcta 2221 atagaatttg atggtgtaca gtctgctgga gatgacagag tacttgtaat gggtgcaact 2281 aataggccac aagagcttga tgaggctgtt ctcaggcgtt tcatcaaacg ggtatatgtg 2341 tctttaccaa atgaggagac aagactactt ttgcttaaaa atctgttatg taaacaagga 2401 agtccattga cccaaaaaga actagcacaa cttgctagaa tgactgatgg atactcagga 2461 agtgacctaa cagctttggc aaaagatgca gcactgggtc ctatccgaga actaaaacca 2521 gaacaggtga agaatatgtc tgccagtgag atgagaaata ttcgattatc tgacttcact 2581 gaatccttga aaaaaataaa acgcagcgtc agccctcaaa ctttagaagc gtacatacgt 2641 tggaacaagg actttggaga taccactgtt taaggaaata cctttgtaaa cctgcagaac 2701 attttactta aaagaggaaa cacaagatcc cccgggctgc aggaattcga tatcaagctt 2761 atcgataccg tcgacctcga gggggggccc ggtaccttaa ttaattaagg taccaggtaa 2821 gtgtacccaa ttcgccctat agtgagtcgt attacaattc actcgatcgc ccttcccaac 2881 agttgcgcag cctgaatggc gaatggagat ccaattttta agtgtataat gtgttaaact 2941 actgattcta attgtttgtg tattttagat tcacagtccc aaggctcatt tcaggcccct 3001 cagtcctcac agtctgttca tgatcataat cagccatacc acatttgtag aggttttact 3061 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt 3121 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 3181 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 3241 tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt 3301 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa 3361 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa 3421 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac 3481 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga 3541 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa 3601 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc 3661 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca 3721 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 3781 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 3841 atcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc 3901 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 3961 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 4021 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 4081 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 4141 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 4201 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 4261 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 4321 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 4381 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt 4441 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 4501 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 4561 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 4621 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 4681 aagccggtct tgtcgatcag gatgatctgg acgaagaaca tcaggggctc gcgccagccg 4741 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg 4801 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 4861 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 4921 ctgaagaact tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 4981 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 5041 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac 5101 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat 5161 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga 5221 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa 5281 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca 5341 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc 5401 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc 5461 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt 5521 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 5581 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 5641 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 5701 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 5761 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa 5821 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 5881 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 5941 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 6001 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 6061 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 6121 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 6181 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 6241 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 6301 atcccctgat tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa 6361 ttacggggtc attagttcat agcccatata tggagttccg //