LOCUS Exported 6577 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression for structure determination; may not be full ORF. ACCESSION . VERSION . KEYWORDS SEC14L4 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6577) TITLE SGC Oxford REFERENCE 2 (bases 1 to 6577) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6577 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." RBS 59..81 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 92..109 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 134..154 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1438..1455 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1508..1526) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1522..1569 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1606..2061 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1693..1712) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1903..1924 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2154..2969) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 2877..2896 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2961..2980 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 3091..3679 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3580..3599 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3833..3850 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3865..4007 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3933..3952) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4093..4115 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4109..4300) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5109..6191) /codon_start=1 /gene="lacI" /product="lac repressor" /label=lacI /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 6153..6172 /label=LacI-R /note="LacI, reverse primer" promoter complement(6192..6269) /gene="lacI" /label=lacI promoter primer_bind 6475..6494 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt 121 agatctgggt accgagaacc tgtacttcca atccatgtcg tcccgtgtgg gtgacctgtc 181 gccgcaacaa caagaagccc tggcccgctt ccgtgaaaac ctgcaagacc tgctgccgat 241 tctgccgaac gcggatgact attttctgct gcgttggctg cgtgcccgca atttcgatct 301 gcagaaaagt gaagacatgc tgcgtcgtca catggaattt cgcaaacagc aggatctgga 361 taacattgtc acctggcagc cgccggaagt gatccaactg tatgattcag gcggtctgtg 421 cggctatgac tacgaaggtt gtccggtgta cttcaatatt atcggcagcc tggacccgaa 481 aggtctgctg ctgagtgctt ccaagcagga catgattcgt aaacgcatca aggtgtgcga 541 actgctgctg catgaatgtg aactgcagac ccaaaaactg ggccgtaaga ttgaaatggc 601 gctgatggtt tttgatatgg aaggtctgag tctgaaacac ctgtggaagc cggcagtgga 661 agtttatcag caatttttct ccatcctgga agctaactac ccggaaacgc tgaaaaatct 721 gattgttatc cgcgcgccga agctgtttcc ggtggccttc aacctggtta aatcattcat 781 gtcggaagaa acccgtcgca aaattgtgat cctgggcgat aattggaaac aggaactgac 841 gaagtttatt agcccggacc aactgccggt tgaatttggc ggcaccatga ccgatccgga 901 cggtaacccg aaatgcctga ccaagatcaa ttatggcggt gaagtgccga aaagctatta 961 cctgtgtgaa caggttcgtc tgcaatacga acacacgcgt tctgttggcc gcggtagctc 1021 tctgcaggtc gaaaacgaaa ttctgtttcc gggttgcgtt ctgcgttggc agttcgcaag 1081 cgatggcggt gacatcggct ttggtgtctt cctgaaaacc aagatgggtg aacagcaatc 1141 agcgcgtgaa atgacggaag tcctgccgtc gcagcgctat aacgcccaca tggtgccgga 1201 agatggctct ctgacctgtc tgcaggcagg tgtctatgtg ctgcgttttg ataataccta 1261 cagtcgcatg cacgcgaaaa agctgtccta cacggttgaa gtcctgctgc cggataaagc 1321 aagcgaagaa accctgcagt ctctgaaagc tatgcgtccg agcccgacgc aatgacagta 1381 aaggtggata cggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac 1441 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct 1501 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg 1561 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg 1621 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 1681 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 1741 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 1801 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 1861 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 1921 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 1981 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 2041 aaatattaac gtttacaatt tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 2101 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa 2161 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat 2221 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg 2281 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat 2341 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc 2401 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta 2461 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga 2521 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac 2581 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct 2641 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga 2701 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg 2761 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct 2821 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg 2881 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag 2941 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca 3001 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 3061 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 3121 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 3181 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 3241 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 3301 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 3361 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 3421 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 3481 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 3541 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 3601 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 3661 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 3721 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 3781 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 3841 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg 3901 tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta 3961 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc 4021 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 4081 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 4141 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct 4201 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa 4261 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg 4321 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg 4381 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat 4441 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag 4501 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg 4561 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc 4621 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta 4681 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg 4741 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct 4801 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc 4861 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga 4921 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca 4981 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg 5041 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg 5101 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 5161 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc 5221 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc 5281 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc 5341 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca 5401 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca 5461 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg 5521 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga 5581 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac 5641 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct 5701 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc 5761 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta 5821 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg 5881 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat 5941 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca 6001 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc 6061 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc 6121 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt 6181 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg 6241 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc 6301 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa 6361 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc 6421 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat 6481 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg 6541 tccggcgtag aggatcgaga tctcgatccc gcgaaat //