LOCUS       sequence-431050-        6878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CDK2-7 chimera in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6878)
  AUTHORS   Mukherjee M, Day PJ, Laverty D, Bueren-Calabuig JA, Woodhead AJ, 
            Griffiths-Jones C, Hiscock S, East C, Boyd S, O'Reilly M
  TITLE     Protein engineering enables a soakable crystal form of human CDK7 
            primed for high-throughput crystallography and structure-based drug 
            design.
  JOURNAL   Structure. 2024 Jun 9:S0969-2126(24)00188-6. doi: 
            10.1016/j.str.2024.05.011.
  PUBMED    38870939
REFERENCE   2  (bases 1 to 6878)
  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"; journalName: "Structure. 
            2024 Jun 9:S0969-2126(24)00188-6. doi: 10.1016/j.str.2024.05.011."
COMMENT     Sequence Label: pET-30a (+)_CDK2-7 chimera
            (I10L,T14Q,V64I,K88...
FEATURES             Location/Qualifiers
     source          1..6878
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1100..1123)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1130..1780)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             complement(1784..1801)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1815..1837
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1852..1876
                     /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     complement(1876..1895)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1877..1895)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1983..2002)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2208..2285
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2286..3368
                     /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(2305..2324)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4177..4368
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4362..4384)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4470..4609
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4525..4544
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4624..4641)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4795..5383)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4875..4894)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5494..5513)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5505..6320
                     /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             5505..6320
                     /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     complement(5578..5597)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6412..6867)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6549..6570)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6761..6780
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 cgacctacag gcgcagatgc ggcaccggtt tggtcacatc ctgaaaaaac ggatgcgcca
      241 gcgccgcttt cgcgctaatg cgtttgttcg gatcataatg cagcatctgg ctcagcaggc
      301 tgcggccatc ttcatccagc ggcggcacca ctttgctaaa atcctggcgc gcccatttcg
      361 gaaagctcgg tttataatcc ggcatgctgg tcacgcccgg ccacaccact tcatccgggg
      421 tgcccagggt gcgaaaaatg cgaaacagct gatcaatttc gctatcgccc ggaaacagcg
      481 cgcggcgggt caccatttcc gcaaaaatgc agcccaggct ccaaatatcc accgcggtgc
      541 tataatattt gcagcccagc agaatttccg gcgcgcgata ccacagggtc accacttcat
      601 gggtataggt gcgcaccggc acgccaaacg cgcgcgccag gccaaaatcc gccagtttaa
      661 tcgcgccttc ggtgttaatc agcaggttgt tcggtttcag atcgcgatgc agcacgcgat
      721 ggctatggca aaacgccagg ccctgcagca gctgaaacag atagctttta atcagcggca
      781 gcggaatgcc ggtcagcgcg ctcgctttca taaacacttc cagatcctga tgcagaaatt
      841 caaacaccag atacagtttg ttttcggtat gaatcacatc cagcagttta ataatgttcg
      901 gatggttcag ttctttcagc aggctaattt cgcgaatcgc ggtgctcggc acgccttcgg
      961 tttcggtatc caggcgaatt tttttcagcg ccaccacttc gccggtcagt ttgttgcgcg
     1021 ctttatacac cacgccatac tggccttcgc ccagtttttc cactttctga aagttttcca
     1081 tgaattccgg ggatcccagg ggcccctgga acagaacttc cagatccgat tttggaggat
     1141 ggtcgccacc accaaacgtg gcttgccagc cctgcaaagg ccatgctata tacttgctgg
     1201 atttcaagta cttatcaatt tgtgggatag cttcaatacg ttttttaaaa caaactaatt
     1261 ttgggaacgc atccaggcac attgggtcca tgtataaaac aacatcaaga gcgtcataca
     1321 acatgaagtc aggatgggtt acatgatcac catttaaata tgttttatga cataaacgat
     1381 cttcgaacat tttcagcatt tcaggtagct tgctaagaaa atcaactttg agagtttcaa
     1441 agtctttact atatgcaatt ctcgaaacac cgtatctaat atccaaaacc gctccttcaa
     1501 gcattgaaat ctctgcacgc tcttttggac aaccacccaa catgttgtgc ttgtcagcta
     1561 tataacgtat gatggccata gactgtgtta atttaacatc accatcaata taataaggaa
     1621 gattgggaaa ctccaaaccc aattcaaact ttttgtttcg ccatttatca ccttcatcgc
     1681 gctcatacaa atgctcttca tatttttctt caagatattc caaaagaagt cgagtgggtt
     1741 gcacaaggcc cttaattttc caataaccta gtatagggga tgcatggtga tggtgatggt
     1801 gtgccatatg tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt
     1861 atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga gatcgatctc
     1921 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     1981 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     2041 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     2101 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     2161 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
     2221 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     2281 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     2341 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     2401 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     2461 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     2521 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     2581 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     2641 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     2701 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     2761 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     2821 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     2881 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     2941 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     3001 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     3061 agtccgggct gcgcgttggt gcggacatct cggtagtggg atacgacgat accgaagaca
     3121 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     3181 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     3241 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     3301 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     3361 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
     3421 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     3481 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     3541 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     3601 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     3661 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc
     3721 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     3781 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     3841 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     3901 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
     3961 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
     4021 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
     4081 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
     4141 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
     4201 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
     4261 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
     4321 taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     4381 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     4441 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     4501 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt
     4561 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     4621 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4681 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4741 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4801 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4861 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4921 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4981 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5041 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5101 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5161 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5221 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5281 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5341 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5401 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa
     5461 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     5521 gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     5581 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga
     5641 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     5701 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     5761 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag
     5821 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     5881 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     5941 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     6001 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     6061 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     6121 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     6181 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     6241 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     6301 tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag
     6361 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     6421 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     6481 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     6541 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     6601 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     6661 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     6721 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     6781 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     6841 cgcttaatgc gccgctacag ggcgcgtccc attcgcca
//
