LOCUS       sequence-273780-        6807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses KinK in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6807)
  AUTHORS   Wang KA, Ng TL, Wang P, Huang Z, Balskus EP, van der Donk WA
  TITLE     Glutamic acid is a carrier for hydrazine during the biosyntheses of 
            fosfazinomycin and kinamycin.
  JOURNAL   Nat Commun. 2018 Sep 11;9(1):3687. doi: 10.1038/s41467-018-06083-7.
  PUBMED    30206228
REFERENCE   2  (bases 1 to 6807)
  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: "Nat 
            Commun."; date: "2018-09-11"; volume: "9(1):3687. doi"; pages: " 
            10.1038/s41467-018-06083-7"
COMMENT     Sequence Label: pET28a-KinK
FEATURES             Location/Qualifiers
     source          1..6807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     103..122
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        103..121
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    122..146
                     /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             161..183
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             202..219
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             229..246
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1774..1791
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1844..1862)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1858..1905
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1942..2397
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2029..2048)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2239..2260
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2489..3304)
                     /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(2489..3304)
                     /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     3212..3231
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3296..3315
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3426..4014
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3915..3934
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4168..4185
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4200..4339
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4265..4284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4425..4447
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4441..4632)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5441..6523)
                     /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     6485..6504
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6524..6601)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6807,1..19)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
       61 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
      121 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
      181 agatatacca tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc
      241 ggcagccata tgtactcccg gtcgtggtcc agccccttct ccgaggcggg cggcacagga
      301 cgtcccgcgt tcgtcaccga gttcggcctg tggaccgacg aacaggccgc tgcggccgag
      361 cagatcgagg cctcgctcga cgagatcgac ctggtccgcc tcgccttcgc cgacccgcac
      421 gggctggccc gctccaagac gctgaccgtg gacgcgttcc gctcggtcct gcgcaacggc
      481 atggacttca gctcgggccc gttcatcttc gacaccggcc acgcggccgc cctggacttc
      541 ctcgccgacc cgggcgtcgg cgtcgacgag atcgcgggcg cgggcagctt cgtgctggtc
      601 ccggacccgc tgacgttcca ggtgctcccc cacgagggac cgcgcaccgc gtgggtgctc
      661 ggtgacgagt atctgcgcga cggctccccg cacccgctct ccgcgcggaa cgtgctgcgc
      721 caggtcgtcg cccggtacgc ggcccgcgac ctcaccccgg tgctcggcct ggaggtcgag
      781 tggtacctga cccgcaagct cgcgggtccg cccgggaacg cgggcaacgg cttcggcctc
      841 cagggcgcgg cccccaaggt cgaggcgatg aactcgggct accagttcaa cctggacgcc
      901 aactacgact cggtggccca cttcaccagc ccgctggcga tgaagctgct cgaactcggc
      961 ctgccgctgc gctcgatcga gcacgagtcg ggcccgggcc agatcgagac gaccttcaac
     1021 ccgatgcacg cgctggacac cgccgacgcg atgctcctgt tccgcaccgt ggtcaagcag
     1081 accgccgcgc gccagggcta ccacgcctcc ttcatggcgc tgccccgcgt cgacagcttc
     1141 gacccgtgcg gctggcatct gcaccagtcg gtgatggaca gcacgaacgg gcgcaacctc
     1201 ttcgcggccg acggcggcgg catctcggac cagggcaagg cgtacatcga cgggctgctc
     1261 tcccgcgccc gcgacctgtg tctgctctcg gtccccaccg tcaacggcta ccgccgcatg
     1321 ggcgcggact tctcgctctc gccgacccgt ctcggctgga gctacgagga ccgcagcgcg
     1381 atgatccggg tggtcggcgg cggcgccggc acgcacatcg agaaccgggt gggcgagccc
     1441 accgccaacc cgtacctcaa catcgccgcc cagctctccg ccgggttcga cggcatggcc
     1501 accgaggtcg gcgcgagcac gcgggagagc ggcgaggagt catacgagac gctgccgcag
     1561 gacctcggcg aggcgctgga ggccttccgg gccggtcagg ccgccgaact gctcggcaag
     1621 ccgctggccg ccaccctggc caagctgaag gagagcgagc tgtcccgcta cgaggcctgg
     1681 cgcgcggccg agcggcccgc cgacggccag gtcaccgagt gggagcagcg cgaatacttc
     1741 gaggccttct gaaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
     1801 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1861 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1921 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1981 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2041 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2101 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2161 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2221 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2281 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2341 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     2401 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2461 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     2521 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     2581 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     2641 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     2701 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     2761 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     2821 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     2881 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2941 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     3001 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     3061 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     3121 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     3181 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     3241 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     3301 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     3361 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3421 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3481 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3541 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3601 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3661 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3721 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3781 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3841 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3901 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3961 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4021 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4081 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4141 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4201 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     4261 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     4321 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4381 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4441 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     4501 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     4561 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     4621 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     4681 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     4741 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     4801 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4861 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4921 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4981 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     5041 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     5101 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     5161 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     5221 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     5281 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     5341 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     5401 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5461 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5521 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     5581 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     5641 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     5701 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     5761 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     5821 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5881 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5941 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     6001 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     6061 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     6121 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     6181 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     6241 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     6301 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     6361 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     6421 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     6481 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     6541 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     6601 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     6661 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     6721 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     6781 aagtggcgag cccgatcttc cccatcg
//
