LOCUS       sequence-274934-        6615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NatL2-R156K in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6615)
  AUTHORS   Song H, Rao C, Deng Z, Yi Y, Naismith JH
  TITLE     The biosynthetic pathway of the benzoxazole in nataxazole proceeds 
            via an unstable ester and has synthetic utility.
  JOURNAL   Angew Chem Int Ed Engl. 2020 Jan 5. doi: 10.1002/anie.201915685.
  PUBMED    31903677
REFERENCE   2  (bases 1 to 6615)
  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: "Angew Chem 
            Int Ed Engl. 2020 Jan 5. doi: 10.1002/anie.201915685."
COMMENT     Sequence Label: pWDY1228
FEATURES             Location/Qualifiers
     source          1..6615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1480..1497
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1550..1568)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1564..1611
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1648..2103
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1735..1754)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1945..1966
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2195..3010)
                     /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(2195..3010)
                     /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     2918..2937
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3002..3021
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3132..3720
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3621..3640
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3874..3891
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3906..4045
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3971..3990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4131..4153
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4147..4338)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5147..6229)
                     /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
                     EDGTRLGVEHLVTLGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6191..6210
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6230..6307)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6513..6532
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg tcgcgctcgc gtccggaact tggtgactgg
      181 tcgagcccgg ccgaactggc cgagctccag cggtcccagc tgccgagggt cctcgcgcag
      241 gcactccgtt cgccgttcta cgcggcccgc taccgcggca ccacaccgcc gcgcaccgcg
      301 gacgacttcg ccggcgtcga ggtgaccgcc aagcaggacc tgcgcgacca gtaccccttc
      361 gggatgctcg cggtcgggcg cgaacacctg gccacctacc acgagtccag cggcaccgcg
      421 ggcgaaccca ccgcctccta ctacacggag gaggactgga ccgacctcgc cgagcgcttc
      481 gcccgcaagt ggaccggcat ccacccctcc gacaccttcc tggtgcgcac cccctacggc
      541 ctggtcatca ccggccacct cgcccaggcc gcgggacggc tgcgcggtgc gaccgtcgtc
      601 cccggcgacg ccaagtcgct ggccaccccg ctctcccgca tggtgcgcgt cctgaagacc
      661 ctcgacgtga cgctcacctg gtgcaacccg accgagatca ccatgctggc cgccgccgcc
      721 aaggccgccg gactccgccc cgaccaggac ttcccgcacc tgcgggccat gttcaccgcc
      781 gccgagccgc tgaccgaggt gcggcgccgc cggctcagcg agatctgggg cggcatcccg
      841 gtcgtcgagg agtacggctc cacggagacc ggcaccatcg ccgggcagtg ccccgagggc
      901 cggatgcacc tgtgggcgga ccgggcgatc ttcgaggtgt acgacccgag gaccggcacg
      961 ctctccgagg cgggccgcgg ccagatggtc gtcaccccgc tgtaccggga cgcgatgccg
     1021 ctgctgcgct acaacctcgc cgacgacgtc gaggtgtcca ccgacccgtg cggctgcggc
     1081 tggctgctgc ccacggtcac cgtgctgggg cgggccggaa ccggtcaccg gatcggtccg
     1141 gcgaccgtca cccagcagcg gctggaggag ctggtgttct ccctgccggc cgcgtacgag
     1201 gtgatgttct ggcgggccaa ggcccacccg gacgtgctgg agctggagtt cgaggcgccc
     1261 gagccggtgc ggcagcgggc cgtcaaggag ctcggcgccg cgctcgaccg cgaactgggg
     1321 gtcccgcacc gcatcaccgg cctcgccccg ggcaccctcg tcccggcgga ggccctcacc
     1381 gcccagcgcg acatcctcaa ggcccgctac ctcttcgcgg aggacgagga ctgggacaag
     1441 gcggtcatgt acttctgaaa gcttgcggcc gcactcgagc accaccacca ccaccactga
     1501 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1561 taactagcat aaccccttgg ggcctctaaa cgggttttga ggggtttttt gctgaaagga
     1621 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1681 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1741 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1801 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1861 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1921 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1981 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2041 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     2101 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2161 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     2221 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     2281 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     2341 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     2401 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     2461 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     2521 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     2581 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     2641 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     2701 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     2761 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     2821 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     2881 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     2941 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     3001 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     3061 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3121 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3181 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3241 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3301 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3361 ccagtgactg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3421 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3481 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3541 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3601 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3661 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3721 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3781 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3841 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3901 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     3961 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     4021 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     4081 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4141 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     4201 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     4261 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     4321 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     4381 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     4441 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     4501 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     4561 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     4621 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     4681 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     4741 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     4801 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     4861 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     4921 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     4981 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     5041 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     5101 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     5161 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5221 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     5281 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     5341 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     5401 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     5461 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     5521 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     5581 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     5641 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     5701 atgtgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     5761 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     5821 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     5881 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     5941 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     6001 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     6061 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     6121 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     6181 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     6241 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     6301 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     6361 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     6421 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     6481 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     6541 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     6601 tcgatcccgc gaaat
//
