LOCUS       sequence-443668-        8562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AzaD in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8562)
  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 8562)
  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-AzaD
FEATURES             Location/Qualifiers
     source          1..8562
                     /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             3434..3451
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3504..3522)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3518..3565
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3602..4057
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3689..3708)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3899..3920
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4149..4964)
                     /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(4149..4964)
                     /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     4872..4891
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4956..4975
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5086..5674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5575..5594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5828..5845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5860..5999
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5925..5944)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6085..6107
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6101..6292)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7101..8183)
                     /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     8145..8164
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8184..8261)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     8467..8486
                     /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 gacactcccg ccgccgtcct
      181 cgccgacctc cgcgaagcgg gcctgtcgct gtccgccgcg ggcggcgacc tcaggctcga
      241 aggccccaag acggccatga cccccgacct cgtcgcgagg ctcaaggccg ccaaggccga
      301 cctcgtcgcg cacctggccg cgttcgagga cgccttcgcg ctgacgcccc tgcaacgcgc
      361 ctacctggtc ggacggtccg gcctgctcga actcggcgac gtcgccaacc aggtctacca
      421 cgagatcgaa ggcgcctggg acctcgaccg actgcgaaag tcccttgagg acaccgcacg
      481 ggcccactcg gcgctgcgac tggccgtcct cgacgaggac cggcaggccg agacccccac
      541 cctgcccggc ttcaccgtaa cggacctgcg cggcctcgac ccgagcgagc gggaccggcg
      601 ccgcgccgcc gtccgcgagc gccgctccca ccgcaggctc ccggccaccg ggcccctcgt
      661 cgatgccgag gtctgcgtcc tcgccgacga ccggatggtc ctgtgcgtca accacgacgg
      721 catggccgtc gacggcatcg gcatgttcct cctcttcggc gaatggcacc gccgatacga
      781 gaccggcggc ggcgacgacg cgccgcggat gcccttccgc gcccacgtgg cggcgatcga
      841 ggccgcggcc gccggccccg cctccgcccg ctcgcgcgag tactggcgct cgcgcctgga
      901 cgaactccct cccgcgcccc ggctgccgct cgccgccgac ccgtccgcga tcggccccac
      961 ccgggtcgtc cagcgccggg tggtgctcga ggccgacgac tggacccgcc tccagcgcga
     1021 gatcaaggcc gccgggctca ccccctcggc cgcgctgctc gccgcctggg ccgaggcact
     1081 gtccctgtgg ggcgccggaa acgcgttcac gctcaacacc accgtctcgc agcgcctccc
     1141 gatccacccc ggcgccttca ccgccgtcgg ccagttctcc gacccgctcc tcgtcgccgt
     1201 ccacatcgat cggacccgga ccttcgccga acgcgcccgc gccctccagg gccgactccg
     1261 caccgacctc gaccaccgcc actactccgg catcgacgtc atgcgcgacc tcgcacgcca
     1321 ccactcggac acccgcgccg ccgccatgcc ctacaccttc aactccacac tggacgccat
     1381 cggcggcgtc gacggctccg cgctcgaatc gttcggcccc gaggtcttca ccgtctccca
     1441 gaccccgcag gtccacctcg acgtgttcgt cctccagcag cacggcgacc tcgtcatccg
     1501 cctcgacgcg gtcgaagcgc tcttcccgcc cggcctgctc gacgccctca ccgccggact
     1561 cgacaccctc ctgcaccgcc tctgcgacgc aaccgcctgg gacgccgaac ggttcgacct
     1621 cctgccggac gcgcagcgcc gcgcccgcaa gcgcgccaac gacaccgagc gggacttcgc
     1681 gcccgccatg ctcaccgacg ccttcctgcg gcacgccgcg cagcgccccg acgcgaccgc
     1741 gatcatcacc gcggacgcgt cgatcaccta cggcgacctc ctcgcccgcg ccgcgggcgc
     1801 cgcgacctgg ctgcgcgaca acggcgccgg ccgcggcgac ctcgtcggcc tggtgatgcg
     1861 gcgcggaccc gagcagatcg tcggcatcct cggcgccctg ctcgccggcg ccgcgtacgt
     1921 cccggtcgac gccgacctcc cctcgacccg ccgcgaccgg ctcctgcgcg acgaccgcct
     1981 cgcccacgtc ctcaccgacg acgacctgga tcacggcctc ccgtcgctcg acctgcgcgc
     2041 ggggccgcgc gcggccgcgg cccccgacgt gcccggcgcc gaccccggcg acctcgccta
     2101 cgtcctctac acctcgggca ccaccggcga acccaagggc gtcatggtga cccaccggaa
     2161 cgtcgccaac gtcgtcgccg actgcgccga gcggttcgcg atcggcccgg acgaccgctt
     2221 cttcgccatc agcgccttca ccttcgacct gtcggtgtgg gacgtcttcg gcgccctgag
     2281 cgccggggcc gcgctggtcg tcccggacgc cgaccgggcc gccgaccccg cgcactggaa
     2341 gacgctcgcc gaagccgccg gagcgacggt gtggaactcg gttccggcga tcgtgcggat
     2401 gctcgccgac cacgacgggt cactgccgcc ctcgctgcga ctggtcatga tgagcggcga
     2461 ccgcatcccg cccgacctgc ccgccgcgct ctaccgcgcc cgacccgacc tgcgcgtcgt
     2521 ctccctcggc gggccgaccg aaacgacgat ctggaacatc tggcatccga tcccgcccga
     2581 ccacccggcg gacgtgccgc tcccgtacgg ccgccccaac gccaacaacc gcgcgcacat
     2641 ccgcgacgcc gacggcaacg actgccccga ctgggtcccc ggcgagatcc tcgccgccgg
     2701 cgccggcgtc acccccggct accggggcgc ccccgacctg accgccgaac gctacgtcga
     2761 cgaccgcggc gaccggctct accgcaccgg cgacctcggc cgctaccggc ccgacggcac
     2821 gatcgacatc ctgggccgca ccgacttcca gatcaaggtc aacggccacc gcatcgaggc
     2881 cggcgaggtc gagacccggc tcgcggccct cgacccggtc gccgaggccg cggtcgtggc
     2941 ccgcaagggc gcctccgggg cggtcctcgt cgcccacctc gtccccgccg ccggccggcc
     3001 cccgctcgcc gagatccgcg aggccctccg cgccgacctg cccgactaca tggtcccggc
     3061 cgcggtcgtg tggcacgacc ggctgccgct caaccgcaac cgcaaggtcg accgggccgc
     3121 gctcgccgac gtcccgatcg ccgaacaggg agccgaagac cgcccgcccg ccgaccccgc
     3181 cgtcgaatcc aggatcgccg cgctgtgggc gagcacgctg cgggtccccg aggtcgaccc
     3241 cgaagccgac ttcaccgccc tcggcggcga ctccctgacc gccgcgcgca tcctgaccgc
     3301 cgtgcgcgcc gagtacggga tcggcctgcc gctggaggcg atcttcgcgc tcaacaccgt
     3361 gcgcgccatg gcgggccacc tcaccgccac caccgaacgg gagcagccgt agaagcttgc
     3421 ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa
     3481 ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     3541 taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg
     3601 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3661 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3721 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3781 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3841 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3901 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3961 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4021 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     4081 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4141 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     4201 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     4261 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     4321 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     4381 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     4441 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     4501 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     4561 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     4621 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     4681 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     4741 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     4801 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     4861 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     4921 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     4981 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     5041 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5101 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5161 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5221 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5281 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5341 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5401 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5461 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5521 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5581 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5641 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5701 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5761 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     5821 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     5881 tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat
     5941 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     6001 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     6061 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     6121 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac
     6181 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
     6241 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
     6301 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
     6361 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
     6421 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
     6481 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
     6541 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
     6601 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
     6661 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
     6721 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa
     6781 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     6841 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     6901 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     6961 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     7021 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     7081 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     7141 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     7201 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     7261 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     7321 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     7381 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     7441 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     7501 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     7561 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     7621 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     7681 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     7741 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     7801 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     7861 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     7921 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     7981 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     8041 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     8101 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     8161 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     8221 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     8281 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     8341 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     8401 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     8461 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     8521 ggccacgatg cgtccggcgt agaggatcga gatctcgatc cc
//
