LOCUS       sequence-273447-        6591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses KinM in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6591)
  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 6591)
  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-KinM
FEATURES             Location/Qualifiers
     source          1..6591
                     /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             1558..1575
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1628..1646)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1642..1689
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1726..2181
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1813..1832)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2023..2044
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2273..3088)
                     /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(2273..3088)
                     /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     2996..3015
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3080..3099
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3210..3798
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3699..3718
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3952..3969
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3984..4123
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4049..4068)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4209..4231
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4225..4416)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5225..6307)
                     /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     6269..6288
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6308..6385)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6591,1..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 tgattccccg ctataccctg cccgcgatgg cggacatctt ctcggaccag
      301 gcgcggtacg cgacctgggt ccgggtggag atcctggctt ccgaggcgca ggcgcgcctg
      361 ggacgggtgc ccgaggacgc ggtcgaggac atgcggcggg ccaaggtccc gacgcccgag
      421 cgggtgcagg agatcgagcg cgagcgcgac cacgaagtgc tctcgttcct cgccgcgtac
      481 tgcgaggaca tcccggacga gtcggcccgc tgggtccacc tcggcatgac cagctacgac
      541 ctcgtcgaca cctcgctggg ctacaacctg gcccgcgcca ccgacctggt gatcgcggcc
      601 gcggtcgagc tgcgcaaggt cctggtcgaa cgggccctgg agcactggga gacggtcatc
      661 gtcggccgca cccacggcgt ccacgccgag ccgacgtcct tcggccacaa gctggcgcag
      721 ttcgcgttcg cggtggaccg ctcgatcacc cggctgcgcg cggcgcgcaa ggccgtggcg
      781 gtgggcacga tctccggctc ggtcggcacg tacgcgctga tcgacccctc cgtcgaggcg
      841 tacgtctgcg aggagctgga cctgggggtg gagccggccc cgagccaggt cgtcgcccgc
      901 gaccggcacg cccagctgat ccaggccgtc gccgtgctcg gcgcgagcgt cgagcagatc
      961 gccctggagc tgcggctgct gcagcgcacc gaggtccgcg aggtcgagga gcagcgcacc
     1021 tcggcgtacc agggctccag cgccatgccc cacaagcgca acccgaccac cagcgagcgt
     1081 ctgacgggtc tggcccggct gctgcgcggt tacgcgacca cggccctgga gaacgtggcg
     1141 ctgtggcacg agcgggatct ggcccaccag tcggtggagc gggtgatcct gccggacgcg
     1201 ctgtcggtgg ggcacttcca ggccacgatg gcggccgacc tggtccgtaa cctcaaggtg
     1261 ttcccggagc ggatgcgggc ggggatcgac cagaccgacg gtcttgtgtt cagctctgcc
     1321 gtactcgccg atctgctggc cgacggggtg gagcgggaga aggcctaccg ggccgtccag
     1381 tccgccgcca accgcaccat cgcgaccggc gagcacttcg gggacacgct gcggcaggag
     1441 ggcatggaca tcggccagct ccgcccggag cgtttcctcg tcaaccacgg tgtgattctc
     1501 aagcgattgg agcagcttcg tgacctggac gactgaaagc ttgcggccgc actcgagcac
     1561 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     1621 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1681 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
     1741 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     1801 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     1861 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     1921 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     1981 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2041 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     2101 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     2161 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2221 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     2281 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     2341 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     2401 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     2461 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     2521 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     2581 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2641 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2701 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2761 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     2821 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2881 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2941 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     3001 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     3061 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     3121 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3181 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3241 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3301 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3361 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3421 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3481 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3541 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3601 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3661 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3721 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3781 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3841 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3901 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3961 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4021 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4081 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     4141 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4201 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4261 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     4321 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4381 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4441 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4501 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4561 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4621 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4681 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4741 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4801 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4861 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     4921 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     4981 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     5041 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     5101 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5161 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5221 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5281 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5341 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5401 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5461 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5521 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5581 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5641 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5701 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     5761 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     5821 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     5881 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     5941 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     6001 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     6061 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6121 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6181 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6241 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6301 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6361 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6421 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6481 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6541 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc g
//
