LOCUS Exported 6888 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression of WW domain #2 from SMURF2. ACCESSION . VERSION . KEYWORDS pHH0103 SMURF2 WW domain #2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6888) TITLE Marius Sudol WW domains JOURNAL Unpublished REFERENCE 2 (bases 1 to 6888) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6888 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..29 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 37..53 /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)." primer_bind 42..64 /label=M13/pUC Reverse /note="In lacZ gene" CDS 138..155 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 171..824 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK" primer_bind 782..804 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 831..854 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 867..884 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 906..926 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" terminator 1083..1130 /label=fd terminator /note="central terminator from bacteriophage fd (Otsuka and Kunisawa, 1982)" rep_origin complement(2723..3178) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2860..2881) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3072..3091 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind complement(3220..3242) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 3328..3468 /label=bom /note="basis of mobility region from pBR322" primer_bind 3383..3402 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(3483..3500) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3654..4241) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(4412..5272) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 5035..5054 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5273..5377) /gene="bla" /label=AmpR promoter primer_bind 5445..5463 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5486..5563 /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." CDS 5564..6646 /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 complement(5583..5602) /label=LacI-R /note="LacI, reverse primer" ORIGIN 1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca 61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga 121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta 181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc 241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca 301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta 361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg 421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat 481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta 541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa 601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat 661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg 721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc 781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc 841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc 901 cggccgaaaa tctgtacttc caaggtcgtc gtgcctctgt ggagctcacc ccgccggatc 961 tgccggaagg ttatgaacaa cgcacgacgc aacaaggtca agtctatttt ctgcatacgc 1021 aaacgggtgt ctcaacctgg catgatccgc gtgtgccgcg cgactaggcg gccgcataat 1081 aaaccgatac aattaaaggc tccttttgga gccttttttt tggagatttt caacgtgaaa 1141 aaattattat tcgcaattcc tttagttgtt cctttctatt ctcactccgc tgaaactgtt 1201 gaaagttgtt tagcaaaacc ccatacagaa aattcattta ctaacgtctg gaaagacgac 1261 aaaactttag atcgttacgc taactatgag ggctgtctgt ggaatgctac aggcgttgta 1321 gtttgtactg gtgacgaaac tcagtgtcta gctagagtgg cggtggctct ggttccggtg 1381 attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg 1441 aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg 1501 ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg 1561 gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt 1621 taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt 1681 ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat 1741 tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt 1801 ttgctaacat actgcgtaat aaggagtctt aatcatgcca gttcttttgg ctagcgccgc 1861 cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac 1921 ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat tggagccaat 1981 caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat atccatcgcg 2041 tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc ctggccacgg 2101 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact 2161 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg 2221 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa 2281 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg 2341 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt 2401 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac 2461 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc 2521 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa 2581 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc 2641 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat 2701 gagctttacc gcaggatccg cgaaattgta agcgttaata ttttgttaaa attcgcgtta 2761 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 2821 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 2881 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 2941 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 3001 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 3061 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 3121 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg 3181 cggatcctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 3241 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 3301 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg 3361 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg 3421 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct 3481 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 3541 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 3601 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 3661 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 3721 ccgacaggac tataaagata ccagggtttc cccctggaag ctccctcgtg cgctctcctg 3781 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 3841 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 3901 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 3961 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 4021 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 4081 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 4141 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 4201 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 4261 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 4321 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 4381 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 4441 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 4501 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 4561 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 4621 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 4681 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg 4741 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 4801 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 4861 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 4921 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 4981 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata 5041 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 5101 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 5161 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 5221 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 5281 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 5341 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 5401 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 5461 ggccctttcg tcttcaagaa ttcccgacac catcgaatgg tgcaaaacct ttcgcggtat 5521 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt 5581 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca 5641 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa 5701 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt 5761 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg 5821 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc 5881 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta 5941 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt 6001 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg 6061 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc 6121 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac 6181 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt 6241 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa 6301 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc 6361 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt 6421 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca 6481 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag 6541 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 6601 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 6661 atgtaagtta gctcactcat taggcacaat tctcatgttt gacagcttat catcgactgc 6721 acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag 6781 gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt 6841 tttgcgccga catcataacg gttctggcaa atattctgaa atgagctg //