LOCUS Exported 6967 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pET-28a-hPKM1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6967) AUTHORS Christofk HR, Vander Heiden MG, Wu N, Asara JM, Cantley LC TITLE Pyruvate kinase M2 is a phosphotyrosine-binding protein. JOURNAL Nature. 2008 Mar 13;452(7184):181-6. doi: 10.1038/nature06667. PUBMED 18337815 REFERENCE 2 (bases 1 to 6967) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6967 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 55..74 /label=T7 /note="T7 promoter, forward primer" promoter 55..73 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 74..98 /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 113..135 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 154..171 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 181..198 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 202..234 /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10" /label=T7 tag (gene 10 leader) /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS 1886..1903 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1956..1974) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1970..2017 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2054..2509 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2141..2160) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2351..2372 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2601..3416) /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 3324..3343 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 3408..3427 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 3538..4126 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4027..4046 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4280..4297 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4312..4451 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4377..4396) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4537..4559 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4553..4744) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5553..6635) /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 6597..6616 /label=LacI-R /note="LacI, reverse primer" promoter complement(6636..6713) /gene="lacI" /label=lacI promoter primer_bind 6919..6938 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac 61 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta 121 actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc 181 ctggtgccgc gcggcagcca tatggctagc atgactggtg gacagcaaat gggtcgcgga 241 tccgaattca tgtcgaagcc ccatagtgaa gccgggactg ccttcattca gacccagcag 301 ctgcacgcag ccatggctga cacattcctg gagcacatgt gccgcctgga cattgattca 361 ccacccatca cagcccggaa cactggcatc atctgtacca ttggcccagc ttcccgatca 421 gtggagacgt tgaaggagat gattaagtct ggaatgaatg tggctcgtct gaacttctct 481 catggaactc atgagtacca tgcggagacc atcaagaatg tgcgcacagc cacggaaagc 541 tttgcttctg accccatcct ctaccggccc gttgctgtgg ctctagacac taaaggacct 601 gagatccgaa ctgggctcat caagggcagc ggcactgcag aggtggagct gaagaaggga 661 gccactctca aaatcacgct ggataacgcc tacatggaaa agtgtgacga gaacatcctg 721 tggctggact acaagaacat ctgcaaggtg gtggaagtgg gcagcaagat ctacgtggat 781 gatgggctta tttctctcca ggtgaagcag aaaggtgccg acttcctggt gacggaggtg 841 gaaaatggtg gctccttggg cagcaagaag ggtgtgaacc ttcctggggc tgctgtggac 901 ttgcctgctg tgtcggagaa ggacatccag gatctgaagt ttggggtcga gcaggatgtt 961 gatatggtgt ttgcgtcatt catccgcaag gcatctgatg tccatgaagt taggaaggtc 1021 ctgggagaga agggaaagaa catcaagatt atcagcaaaa tcgagaatca tgagggggtt 1081 cggaggtttg atgaaatcct ggaggccagt gatgggatca tggtggctcg tggtgatcta 1141 ggcattgaga ttcctgcaga gaaggtcttc cttgctcaga agatgatgat tggacggtgc 1201 aaccgagctg ggaagcctgt catctgtgct actcagatgc tggagagcat gatcaagaag 1261 ccccgcccca ctcgggctga aggcagtgat gtggccaatg cagtcctgga tggagccgac 1321 tgcatcatgc tgtctggaga aacagccaaa ggggactatc ctctggaggc tgtgcgcatg 1381 cagcacctga tagctcgtga ggctgaggca gccatgttcc accgcaagct gtttgaagaa 1441 cttgtgcgag cctcaagtca ctccacagac ctcatggaag ccatggccat gggcagcgtg 1501 gaggcttctt ataagtgttt agcagcagct ttgatagttc tgacggagtc tggcaggtct 1561 gctcaccagg tggccagata ccgcccacgt gcccccatca ttgctgtgac ccggaatccc 1621 cagacagctc gtcaggccca cctgtaccgt ggcatcttcc ctgtgctgtg caaggaccca 1681 gtccaggagg cctgggctga ggacgtggac ctccgggtga actttgccat gaatgttggc 1741 aaggcccgag gcttcttcaa gaagggagat gtggtcattg tgctgaccgg atggcgccct 1801 ggctccggct tcaccaacac catgcgtgtt gttcctgtgc cgtgagaatt cgagctccgt 1861 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa 1921 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc 1981 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg 2041 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 2101 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 2161 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 2221 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 2281 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 2341 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 2401 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 2461 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt 2521 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 2581 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta 2641 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa 2701 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact 2761 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag 2821 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc 2881 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa 2941 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga 3001 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata 3061 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca 3121 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc 3181 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta 3241 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt 3301 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc 3361 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca 3421 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta 3481 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 3541 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 3601 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 3661 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 3721 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 3781 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 3841 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 3901 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 3961 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 4021 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 4081 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 4141 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 4201 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 4261 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg 4321 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca 4381 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg 4441 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 4501 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 4561 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt 4621 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa 4681 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg 4741 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac 4801 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt 4861 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc 4921 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg 4981 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg 5041 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc 5101 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc 5161 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca 5221 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg 5281 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc 5341 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga 5401 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc 5461 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat 5521 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 5581 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 5641 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc 5701 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa 5761 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat 5821 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg 5881 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc 5941 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc 6001 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag 6061 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc 6121 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg 6181 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca 6241 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg 6301 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg 6361 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg 6421 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc 6481 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc 6541 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca 6601 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct 6661 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc 6721 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc 6781 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc 6841 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg 6901 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc 6961 gccggtg //