LOCUS Exported 6150 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses mature human Transforming Growth Factor alpha fused to SPM in bacterial cells for calcium-induced covalent coupling to Epidermal Growth Factor Receptor.. ACCESSION . VERSION . KEYWORDS pET28a-TGF-alpha-GSY-SPM SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6150) AUTHORS Scheu AHA, Lim SYT, Metzner FJ, Mohammed S, Howarth M. TITLE NeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins. JOURNAL Nat Commun 12, 717 (2021). REFERENCE 2 (bases 1 to 6150) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6150 /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 991..1008 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1085..1103) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1099..1146 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1183..1611 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1270..1289) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1480..1501 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1730..2545) /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 2453..2472 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2537..2556 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2667..3255 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3156..3175 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3409..3426 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3441..3580 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3506..3525) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3666..3688 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3682..3873) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4682..5764) /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 5726..5745 /label=LacI-R /note="LacI, reverse primer" promoter complement(5765..5842) /gene="lacI" /label=lacI promoter primer_bind 6048..6067 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atataccatg gtcgtaagcc attttaatga ctgtcctgac 121 tcacacaccc agttctgttt tcacggcact tgtcgcttct tagtgcaaga agacaagcca 181 gcgtgtgtct gccatagtgg gtatgttggg gctcgttgcg aacatgctga ccttcttgcc 241 ggctcgtatg atccactggc attagatctt gatggcgacg gtatcgaaac ggtggcaacg 301 aaggggtttg ccggctcctt attcgatcac acaaataacg gaattcgcac agctactgga 361 tgggtaagtg ccgacgacgg cctgttggtg cgcgacctga atggcaacgg gattatcgac 421 aatggcgcgg aactttttgg agataacacg aaattggcag acgggagttt tgctaagcac 481 gggtatgcgg cgttggcgga gttggatagt aacggagaca atatcatcaa tgcggcagat 541 gcggcattcc agactttgcg cgtgtggcaa gatctgaatc aggacggtat ttcccaagct 601 aatgagcttc gtacattgga ggagcttggt atccagtcgt tagacctggc atacaaagat 661 gtcaacaaga atttaggtaa cggtaatact ttagctcagc aaggtagcta caccaaaacc 721 gacggaacca ctgcaaaaat gggcgacttg ttgctggcag cagataatct gcactcgcgt 781 ttcaaggaca aagtagaact tactgcggaa caggcaaagg ccgcaaatct ggccggtatt 841 ggtcgtttac gcgacttacg cgaggccgca gctctttctg gagatctggc taatatgtta 901 aaagcctatt cagccgcgga gaccaaagaa gcccaattgg ctctgttgga taatttgatt 961 cacaaatggg ccgagacgga cggcagcagc catcatcacc atcaccacag cagcggcgag 1021 ccagaggctt aataagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct 1081 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt 1141 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgscc tgtagcggcg 1201 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 1261 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 1321 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 1381 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 1441 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 1501 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 1561 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 1621 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 1681 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact 1741 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt 1801 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa 1861 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc 1921 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg 1981 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct 2041 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga 2101 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc 2161 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata 2221 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac 2281 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca 2341 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg 2401 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag 2461 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag 2521 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca 2581 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 2641 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 2701 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 2761 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 2821 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 2881 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 2941 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 3001 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 3061 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 3121 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 3181 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 3241 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 3301 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 3361 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 3421 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 3481 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 3541 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc 3601 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 3661 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca 3721 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc 3781 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc 3841 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct 3901 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga 3961 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg 4021 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg 4081 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg 4141 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt 4201 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga 4261 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag 4321 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa 4381 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac 4441 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac 4501 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc 4561 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa 4621 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg 4681 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 4741 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg 4801 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt 4861 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat 4921 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca 4981 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca 5041 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg 5101 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat 5161 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga 5221 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg 5281 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat 5341 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca 5401 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc 5461 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg 5521 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca 5581 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca 5641 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc 5701 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt 5761 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg 5821 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt 5881 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca 5941 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg 6001 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg 6061 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg 6121 tagaggatcg agatctcgat cccgcgaaat //