LOCUS       sequence-443664-        7542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AzaE in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7542)
  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 7542)
  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-AzaE
FEATURES             Location/Qualifiers
     source          1..7542
                     /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             2414..2431
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2484..2502)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2498..2545
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2582..3037
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2669..2688)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2879..2900
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3129..3944)
                     /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(3129..3944)
                     /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     3852..3871
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3936..3955
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4066..4654
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4555..4574
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4808..4825
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4840..4979
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4905..4924)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5065..5087
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5081..5272)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6081..7163)
                     /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     7125..7144
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7164..7241)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7447..7466
                     /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 ccatatgatc ctgaaccact acagcgaatc
      181 cgatctctcc gaggccttcg gcatcgacat gtcctcagta gacggactcg gcgtcggcgc
      241 cggctggggc cgcgtcgcgc ccggcgcgag ctccaccagc caccagcacg acgagaccga
      301 gttcttcgtc atcgtctcgg gcgaaggcga gctccgcatc gacggcgcgg tccatccggc
      361 ccggcccggc accgtggtcc tgttcgagcc cttcgaatcc cacgtcatca ccaacaccgg
      421 cgaggccgac ctggtcttct tcacccagta ctggcgcgac gccgcccgcg cccggcacgc
      481 cgtcgcctcc cggacccgga cccgcgccgc cgggcgcccc cagttcgtgt tctccacccc
      541 gccgaccccc aacggcgacc tccacctggg acacctctcg gggccctacc tcggcgccga
      601 cgcctacgtg cgcttccagc ggctgaacgg caaccgggcc tggcacctca ccggcagcga
      661 cgactaccag agctatgtgg aggccgccgc cgagcgcgac ggcgtcaccc ccgccgaggc
      721 cgcccggcac tactccgccg agatcgccgc caccctcaag gccatggaca tcgacgtcga
      781 cgagtactac acgacgagta cggacgacgg ctacgaggaa ggcctgcggc gcttcttcac
      841 ccgcctggtc cgctccgggt ccgtccgccc gaaggccact cccgcgctgg tcgaaggcga
      901 gggcggcgag tacctgtacg aagtggacgt ctcggggacc tgcccggggt gctcgggtcc
      961 cactggcggc aacatctgcg aggagtgcgg cgagcccaat ctggtcgccg acctcggcga
     1021 gccgcgctcg cgccgcggcg acgccgcccc gcgggcggcc gaggtcgagc gctggaccct
     1081 gccgctccac gagttcgccg agaccgtcgc gggccaccac ctgctcgggc gcgtcccggc
     1141 ccgcctgcgc gaactggccc accgggtctt cgcccgggac ggcctcgaac tgcccgtctc
     1201 ccacccgtcc gactggggcg tgcgcccggc cgaggacgac ctcgcctccc aggtcatctg
     1261 ggtgtggccc gagatgagct acggcttcct ccacggcatc gagcgcctgg gcgccgggct
     1321 cggggagcag tggtcgcgcg agcggccctc cccggactgg aaggtcgtcc acttcttcgg
     1381 ctacgacaac agcttctacc actcgatcct ctacccggtc ctgtaccggc tcgcgttccc
     1441 ccagtggaac cccgacatcg actaccacgt caacgagttc tacctgctcg aaggcgcgaa
     1501 gttctccacc agccgccgcc acgccatctg gggcaaggag atcctgagcc ccgagaccgt
     1561 ggacgcggtc cgctactacc tgtgccgcac ccgtcccgag gacgagcgca ccgacttccg
     1621 ccgcgccgag tacgaccgcg ccgtcaccga tgtcctcatc ggacagtggc aggcctggct
     1681 ggacgacctc ggcgagcgcg tccgcagcga ccacggcggc ctcgtccccg acgccgggac
     1741 ctggacgacc gagcacgcgg acttcctcgc gcggctcacc caccgcctcg agcaggccac
     1801 cgcggccctg ggcgccgaag gcttctcgct ccgccgggcc gcggcggtcc tcgacgccct
     1861 ggtcgaggac gcgacggcgt tctcccggct ccagtcgcgc ctgcgcggcc tcgaccgctg
     1921 gtcctccgag gcgcgcaccg cgaccgccct cgaactcgcc gccgcccgcc tgctcgcgac
     1981 cgccgccgcg cccctgatgc cgcgcttctc gcagcggctc gccgcatcgc tcgggctcgc
     2041 ggtcgacgcc tggcccgacg cggtcgagct cgtcccgccc ggcaaccgga ccacattgga
     2101 ctcgacccgc ttcttcgcgg cccccgcgac acccgcttcc ctcccgtggc tgcggtccaa
     2161 ggtcgccgtc ctgctcggcc tcgaagcgga cttcgcggac ctgtcgctgc ccgcactcgg
     2221 cgcgaactcg ctggccgcga ccaccctcca gtaccacgcg ctcaccgagc tcaaggccga
     2281 cgtcccgctg gagaccctcc tcgccgacgc gcccctgagc gcgctcgccg ccgcactgga
     2341 cggcatggcc gacgccgacc tgctcgccgc actcggaggt gagacccgat gaaagcttgc
     2401 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     2461 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     2521 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     2581 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2641 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2701 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2761 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     2821 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2881 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     2941 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3001 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     3061 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3121 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     3181 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     3241 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     3301 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     3361 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     3421 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     3481 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     3541 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     3601 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     3661 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     3721 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     3781 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     3841 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     3901 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     3961 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     4021 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4081 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4141 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4201 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4261 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4321 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4381 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4441 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4501 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4561 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4621 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4681 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4741 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4801 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4861 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     4921 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     4981 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5041 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5101 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     5161 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     5221 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     5281 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     5341 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     5401 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     5461 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     5521 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     5581 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     5641 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     5701 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     5761 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     5821 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     5881 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     5941 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     6001 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     6061 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6121 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     6181 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     6241 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     6301 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     6361 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     6421 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     6481 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     6541 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     6601 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     6661 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     6721 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     6781 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     6841 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     6901 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     6961 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     7021 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     7081 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     7141 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     7201 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     7261 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     7321 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     7381 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     7441 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     7501 ggccacgatg cgtccggcgt agaggatcga gatctcgatc cc
//
