LOCUS       sequence-443667-        5922 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AzaB in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5922)
  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 5922)
  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-AzaB
FEATURES             Location/Qualifiers
     source          1..5922
                     /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             794..811
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(864..882)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      878..925
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      962..1417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1049..1068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1259..1280
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1509..2324)
                     /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(1509..2324)
                     /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     2232..2251
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2316..2335
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2446..3034
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2935..2954
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3188..3205
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3220..3359
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3285..3304)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3445..3467
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3461..3652)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4461..5543)
                     /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     5505..5524
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5544..5621)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5827..5846
                     /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 gacaccgcct ggaacgagga
      181 cgcacccgcc gtcctcgaga acgaccacgt ccgcctgcgg cccgtgaccg aggacgaccg
      241 cgaaccgctg cgcaagatcg ccttcggcga gggcatctgg gactacttcg tcagccgcgt
      301 cgactccgac gcggacttcg acgcgttctt cgacgccatg ctcgccgacc acgccgccgg
      361 gcgccgcgcc gtcttcgtca tcgtcgacaa gggcgccgac cgggccgccg gcagctccag
      421 ctacggcaac atggccgagg ccgaccggcg cctggagatc ggctggtcct ggctcggcgt
      481 ggagttccag ggccgcggcg tcaaccgctg ggccaagtac ctgctgctcc agcacgcctt
      541 cgaggacctg gacgccgtcc gcgtggagtt caagaccgac gtgctcaacg agcgcgcccg
      601 cgcgggcctg cgcaagatcg gcgcccgcga ggagggcgtg ctgcgcagct tcaaccccat
      661 gccgggcggc cgcaggcggg acgccgtcta ctactccgtg ctcgcgggcg aatggccgtc
      721 ggtgaagacg cagctccgcg accgcggcaa ggccgtgaac ccgtcgctgt gaaagcttgc
      781 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
      841 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
      901 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
      961 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1021 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1081 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1141 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1201 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1261 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     1321 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     1381 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     1441 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1501 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     1561 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     1621 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     1681 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     1741 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     1801 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     1861 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     1921 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     1981 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     2041 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     2101 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     2161 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     2221 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     2281 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     2341 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     2401 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2461 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2521 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2581 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2641 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2701 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2761 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2821 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2881 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2941 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3001 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3061 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3121 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3181 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3241 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     3301 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     3361 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3421 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3481 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     3541 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     3601 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     3661 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     3721 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     3781 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     3841 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     3901 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     3961 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     4021 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     4081 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     4141 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4201 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4261 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4321 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4381 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4441 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4501 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4561 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4621 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     4681 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     4741 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     4801 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     4861 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     4921 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     4981 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5041 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5101 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5161 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5221 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5281 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5341 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5401 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5461 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5521 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     5581 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     5641 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     5701 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     5761 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     5821 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     5881 ggccacgatg cgtccggcgt agaggatcga gatctcgatc cc
//
