LOCUS       sequence-443666-        6639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AzaG in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6639)
  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 6639)
  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-AzaG
FEATURES             Location/Qualifiers
     source          1..6639
                     /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             1511..1528
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1581..1599)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1595..1642
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1679..2134
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1766..1785)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1976..1997
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2226..3041)
                     /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(2226..3041)
                     /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     2949..2968
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3033..3052
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3163..3751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3652..3671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3905..3922
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3937..4076
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4002..4021)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4162..4184
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4178..4369)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5178..6260)
                     /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     6222..6241
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6261..6338)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6544..6563
                     /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 ccatatggga accgcacctc agcaccattc
      181 gacatccgat gatccgacgc cccacctccg gatcgtcggc atcggcgccg gcccggccaa
      241 cctcagcctg gcggccctgc tgcacggcga cgccgcgcag ccgcaccggt tcttcgaccg
      301 caaggccgac ttcacctggc acgacgacca gctcatcacc ggcgcgaccc tccaggtgtc
      361 gatcttcaag gacctggtga gcctctccga cccgaccaac cggttctcgt tcctgtccta
      421 cctccacgag cacgggcgga cctaccactt cctgaacgcc aagttcgccg aggtgccgcg
      481 caccgagttc cgcaactacc tggcctgggc cgcgcggagc aacgagaaca tcgccttcgg
      541 cgaatcggtc gagcgcgtcg acttcgacgg ggtcttccac gtcaccaccg accggcgccg
      601 cgtcacggcc gacaacctcg ccgtcggcgt cggcaccgtc ccgtgggtcc ccgacttcgt
      661 ccggccccac cggggcccca cccagttcca cgtcagcgag cacctgagcc gctcgaaggc
      721 cctgggcggc aagcgcgtcg ccgtcgtcgg cggcggccag tccggcgccg aggcgttcct
      781 ggacctgctc tcgcggcccg aatccgaacg gccccagcgc gtcgtgtgga tctcccggcg
      841 ccgcaactac ttcccgatcg acgactcgac cttcaccaac gacttcttca tgcccgacta
      901 ctccgactac ttcttccggc tgcccgaggc caagcggtcc gagctcatct cggcccacct
      961 gctcgcctcg gacggcatct cggagtccac gctccgcgcg atctaccagg agctgtaccg
     1021 gctccagttc atcgagcgcg acgagcaccg gttcgggctg ctgcccaacc ggaccgtcac
     1081 cgccgccgac agggccggag gcggcgcgtg ggagctgacc ctgcaccaca acgacgagcc
     1141 cgaccagacc cagtgcctcg cggtcgacgc ggtcgtgtgg gccaccggct tccggcccgc
     1201 cccgaagccg ttcctggaac cgctgctcga ccgcatggcg accgagcacg gcgagttccg
     1261 cgtggacgag gacttcgcgg ccgagtggga cggcccgccg gaccggagca tcttcgtgca
     1321 gaacgcggtg cgcggccagc gcggcctgcc cgacgtcaac ctgagcctca acgcctggcg
     1381 ggcccggcgc atcgccgacc gcctgcgcgg gcgccccgcc gccgacccgg cgatgtcgtt
     1441 catcgagtgg tcggccaagc acgccaggga gacgcagtgg acgctgtaga agcttgcggc
     1501 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     1561 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1621 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     1681 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1741 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1801 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1861 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1921 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1981 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     2041 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     2101 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     2161 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     2221 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2281 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2341 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2401 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2461 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     2521 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2581 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2641 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2701 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2761 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2821 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2881 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2941 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     3001 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     3061 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     3121 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3181 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3241 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3301 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3361 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3421 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3481 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3541 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3601 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3661 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3721 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3781 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3841 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3901 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3961 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     4021 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     4081 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4141 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4201 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     4261 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     4321 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     4381 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     4441 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4501 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4561 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4621 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4681 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4741 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4801 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     4861 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     4921 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     4981 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     5041 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     5101 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     5161 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5221 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     5281 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     5341 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     5401 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     5461 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     5521 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     5581 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     5641 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     5701 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     5761 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5821 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5881 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5941 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     6001 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     6061 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     6121 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     6181 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     6241 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6301 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     6361 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     6421 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     6481 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     6541 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     6601 cacgatgcgt ccggcgtaga ggatcgagat ctcgatccc
//
