LOCUS       sequence-151378-        6967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Nature"; date: "2008-03-13- 13"; volume: "452"; issue: "7184"; 
            pages: "181-6"
COMMENT     Sequence Label: pET-28a-hPKM1
FEATURES             Location/Qualifiers
     source          1..6967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     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
//
