LOCUS       sequence-443671-        6474 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AzaM in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6474)
  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 6474)
  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-AzaM
FEATURES             Location/Qualifiers
     source          1..6474
                     /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             1346..1363
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1416..1434)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1430..1477
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1514..1969
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1601..1620)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1811..1832
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2061..2876)
                     /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(2061..2876)
                     /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     2784..2803
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2868..2887
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2998..3586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3487..3506
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3740..3757
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3772..3911
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3837..3856)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3997..4019
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4013..4204)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5013..6095)
                     /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     6057..6076
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6096..6173)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6379..6398
                     /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 cctgccaccg ctttcgaccc
      181 cgacgtgctg ggcctgccgc tgttcgagga ccggcaccgc gacctcgccg aacgcctcga
      241 ccgctggtgc tccggccgcg actgggccgg cccggccggc cccgccgacc ggtgccgcgc
      301 gctgctggcc gagatcggcg cggccggatg gctgcgccac ctcgacaccg accgccccgc
      361 aacggaactg cggtcgctgt gcgtccggcg ccaggtcctc gcctaccacg aggacctcgc
      421 cgactacgtc tactccattc aggagctgtg cgccgccgcg gtcacccgct acggcagcgg
      481 cgagcagcgc cgccgccacc tgccgggcct cgccgccggg accggcatcg gggcgctcgc
      541 gctctccgaa ccgggcgcgg gctccgacct cgccgccgtc gcgctcgagg ccgcgcccga
      601 cggcgacggc ttcgtcctca acggcgccaa gacctggatc gcccagggcg acatcgcgga
      661 cgtgctcgtc gtgctcgccc gcaccggcga cggccccggc ccgctcgggc tcaccgcgtt
      721 cctcgtcccc ggcgacgccc ccggcctcaa ggcctcgccg atcggcgcca tcgcgccccg
      781 gtcgtgggcc gagatcgcct ttaccgactg ccgcgtcggt ccggacgcgg tgctcggcga
      841 acgcggacgc ggctacgtgg tcgccctcga cgtgctcgag cgggcccgca tgaccgtcgc
      901 cgccgccgcg gtcggcttcg ccgcccgcgc cttccgcctc gcgctcgacc acgcccgcgg
      961 ccgcagggcc ttcggaagcc gcctggccga cctccagctc gtcaagtcct cgctcgcgga
     1021 catgcgcgtc aagctgtcgg cggcgtcgct gctgaccgcc cgcgccgcct gggccgccga
     1081 cagcggcgag gactacgccg aggcctcctc gaccgcgaag gtctacgcca ccgaggccgc
     1141 cggcgagatc gtcgacgcgg ccgtccagat cttcggcgcg gccgggctcg tcgccgacag
     1201 ccccgtggag cggctgtacc gccaggtccg ctccctgcgc atctacgaag gaccgtccga
     1261 ggtgctccag atgagcatcg ccgaatccct gtgaccgacg acgaccgacc gggaggcccc
     1321 atagaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
     1381 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     1441 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     1501 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1561 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1621 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1681 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1741 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1801 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1861 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1921 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     1981 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2041 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     2101 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     2161 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     2221 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     2281 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     2341 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     2401 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     2461 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     2521 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     2581 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     2641 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     2701 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     2761 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     2821 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     2881 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     2941 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3001 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3061 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3121 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     3181 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3241 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3301 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3361 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3421 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3481 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3541 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3601 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     3661 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     3721 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     3781 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     3841 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     3901 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3961 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4021 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     4081 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     4141 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     4201 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     4261 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     4321 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     4381 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     4441 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4501 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4561 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4621 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     4681 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4741 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4801 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4861 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4921 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     4981 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5041 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5101 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     5161 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     5221 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     5281 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     5341 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     5401 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5461 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5521 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5581 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5641 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5701 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5761 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5821 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5881 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     5941 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     6001 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     6061 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     6121 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     6181 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     6241 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     6301 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     6361 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     6421 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tccc
//
