LOCUS       sequence-273443-        5709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses KinN in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5709)
  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 5709)
  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-KinN
FEATURES             Location/Qualifiers
     source          1..5709
                     /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"
     regulatory      367..376
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             676..693
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(746..764)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      760..807
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      844..1299
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(931..950)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1141..1162
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1391..2206)
                     /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(1391..2206)
                     /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     2114..2133
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2198..2217
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2328..2916
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2817..2836
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3070..3087
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3102..3241
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3167..3186)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3327..3349
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3343..3534)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4343..5425)
                     /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     5387..5406
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5426..5503)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(5709,1..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 tgacctggac gactgagcgg gtggagggcg ccgacctgga cctggaggcg
      301 gtcctcgacg tctaccgcag ctccgggctc ggcgagcgcc gtcccatcga ggacgtggag
      361 cggttcgccg ccatggtccg caacgccaac ctcgtggtgg tggcgcggga cgcggagggc
      421 aggctcatcg gcatcgcccg cagcatctcc gacttctcct acgcgacgta cctctcggac
      481 atcgcggtga gcggcgacta ccagcgctcg ggcatcggcc gcgcgctcat cgacgccacg
      541 cagaaggagg ccccgcaggc gaagatcatt ctcctgtcgg cgccggcggc ggtggagtac
      601 tacccgcaca tcggcttcac ccagcacaac tccgcctggg tgctcaaccc gtagaagctt
      661 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
      721 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
      781 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
      841 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
      901 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      961 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1021 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1081 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1141 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1201 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1261 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1321 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1381 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     1441 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     1501 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     1561 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     1621 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     1681 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     1741 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     1801 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     1861 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     1921 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     1981 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2041 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2101 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     2161 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     2221 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     2281 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2341 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2401 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2461 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2521 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2581 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2641 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2701 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2761 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     2821 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     2881 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     2941 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3001 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3061 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     3121 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3181 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3241 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3301 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3361 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     3421 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     3481 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     3541 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     3601 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     3661 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     3721 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     3781 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     3841 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     3901 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     3961 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     4021 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4081 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4141 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4201 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4261 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4321 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4381 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     4441 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     4501 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     4561 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     4621 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     4681 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     4741 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     4801 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     4861 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     4921 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     4981 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5041 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5101 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5161 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5221 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5281 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5341 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     5401 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     5461 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     5521 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     5581 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     5641 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     5701 tccccatcg
//
