LOCUS       sequence-443670-        6822 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AzaC in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6822)
  AUTHORS   Van Cura D, Ng TL, Huang J, Hager H, Hartwig JF, Keasling JD, 
            Balskus EP
  TITLE     Discovery of the Azaserine Biosynthetic Pathway Uncovers a 
            Biological Route for alpha-Diazoester Production.
  JOURNAL   Angew Chem Int Ed Engl. 2023 Jul 10;62(28):e202304646. doi: 
            10.1002/anie.202304646. Epub 2023 May 31.
  PUBMED    37151182
REFERENCE   2  (bases 1 to 6822)
  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."; date: "2023-07-10"; volume: "62(28):e202304646. doi";
            pages: " 10.1002/anie.202304646. Epub 2023 May 31"
COMMENT     Sequence Label: pET28a-AzaC
FEATURES             Location/Qualifiers
     source          1..6822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..27
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8..26
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    27..51
                     /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             66..88
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             107..124
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..151
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1694..1711
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1764..1782)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1778..1825
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1862..2317
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1949..1968)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2159..2180
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2409..3224)
                     /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(2409..3224)
                     /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     3132..3151
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3216..3235
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3346..3934
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3835..3854
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4088..4105
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4120..4259
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4185..4204)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4345..4367
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4361..4552)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5361..6443)
                     /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     6405..6424
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6444..6521)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6727..6746
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
       61 ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc atcatcatca
      121 tcacagcagc ggcctggtgc cgcgcggcag ccatatgacc gcatcgatcc tcaaccgcat
      181 catcgccgaa cgtcccgccc acgacggcgt cctcaccgtc gccgggctcg gccaccgccg
      241 cgccatgccg ctcgccgaac tccacgaccg cgcccgagcc gtcgccgggg ccctgcgacg
      301 aggcggcatc ggcgagggcg accgcatcgg cgtcctcgcc gccaaccgca tcgagtggat
      361 cctgctcgat ctcgccgcgc tcatgctcaa ggcccagacc gccgggttcg aacccggcaa
      421 gttcacgccg gacgccgaac tggtcgaacg gtacgggctc gaccagctct acaccgacgc
      481 cccgatcccc gacggcgcgc cgcccgcagt gcgcccgatc gccgagatcg cgcggcacgc
      541 cgacgaggac ccgcccgaga tccatgcgcc cctttgggaa ccggaggact gcaccaccgt
      601 caagttcacc tccggctcca ccggagtccc caaaggactc gaagccacgg tcgccagcat
      661 cgactcctcg ctgcgcgacg tccagggcat gttcgaccac cgccccggcg accggctcct
      721 ggtgttcctg ccgctgtcgc tgctccagca gcgctactgg ctctactcgg cgctgcgctg
      781 gggccacgac ctcgtcgtca cgacctacca gtccgtgttc gccgtgatgc ccgccgtcga
      841 gccgaccgtc gtcatggccg tccccgggtt cttcgactcc gccaggcgcc acctcgaagg
      901 acggctcgcc gacggcgtcg acgccgccaa ggccgccgcc gagctgttcg gctcccgcat
      961 ccgctacctg tggaccggct cggcccccgc cgacccggcc atgctccgct tcttcaccga
     1021 ggccggcctg cccatcttcg aaggctacgg cctcaacgag acctgcatcg ccaccaagaa
     1081 ccaccccggc gcccaccgcg aaggcagcgt cggcaaggcc ctgcccggca aggagatcct
     1141 catcgacgcc gacggcgtcg tgtgcgtccg cagcgaccac cccgtcaaca ccgcctacac
     1201 cttcgcggcg ccaggcgaga gcgagcggat gttcaagccc ggcgggctcg tccgcaccgg
     1261 cgacctcggc cacctcgacg aggacggcta cctgtacatc cacggccgca gcgacgacgt
     1321 catcgtcctc gacaacggca agaaggtcgt cgtccgcccg atcgagaccc gcttccgcga
     1381 caccggcgcc gtcgccgaat gcgtcctata ctgccgcaac cagaccgggc tcgtcgccgt
     1441 cgtctccccg catcccgggc ccgtcgaccg cgccgccgtc gcggccgcgc tcgccgcggt
     1501 caacgccggc tccgaacccg acgagcggat ctcccgcgtc atcaccgccg acgagccctt
     1561 caccatcgac aacggactcc tcacctccca gttcaaaccc atccgggggc gcatcctcga
     1621 gcgctaccgc gaccgcatcg ccgacccgaa agaaggactc catgcccgct gaaagcttgc
     1681 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     1741 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     1801 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     1861 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1921 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1981 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2041 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2101 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2161 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2221 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     2281 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     2341 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     2401 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     2461 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     2521 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     2581 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     2641 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     2701 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     2761 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     2821 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     2881 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2941 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     3001 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     3061 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     3121 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     3181 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     3241 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     3301 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3361 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3421 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3481 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3541 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3601 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3661 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3721 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3781 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3841 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3901 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3961 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4021 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4081 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4141 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     4201 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4261 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4321 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4381 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     4441 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     4501 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     4561 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     4621 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     4681 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     4741 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     4801 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     4861 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4921 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4981 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     5041 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     5101 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     5161 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     5221 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     5281 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     5341 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5401 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     5461 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5521 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5581 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5641 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5701 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5761 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5821 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5881 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5941 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     6001 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     6061 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     6121 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     6181 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     6241 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     6301 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     6361 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     6421 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     6481 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6541 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6601 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     6661 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6721 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6781 ggccacgatg cgtccggcgt agaggatcga gatctcgatc cc
//
