LOCUS       sequence-458637-        5568 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express AzaQ in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5568)
  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 5568)
  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-AzaQ
FEATURES             Location/Qualifiers
     source          1..5568
                     /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             440..457
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(510..528)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      524..571
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      608..1063
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(695..714)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     905..926
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1155..1970)
                     /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(1155..1970)
                     /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     1878..1897
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1962..1981
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2092..2680
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2581..2600
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2834..2851
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2866..3005
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2931..2950)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3091..3113
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3107..3298)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4107..5189)
                     /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     5151..5170
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5190..5267)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5473..5492
                     /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 ccatatgccc gctgacctcg acaccctggc
      181 ccgcgaacag ctcgcgcagg tcctgcgcga ccggcccgac ccggccaccc tcgacgccgg
      241 tgaggacctc gcggagacct acgggctcac ctcgctcaac aaggtcctgt tcctcacctc
      301 gctgtgccgg gccgccgacg tcggcctcga gcgcttcacc gaggacgacc tcgcccggat
      361 gcgcacgctc ggcgacgtcg tcgaggccat gcgtccccac gccgaccaga agggatgaaa
      421 gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
      481 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
      541 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
      601 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      661 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      721 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      781 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      841 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      901 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      961 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     1021 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     1081 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1141 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     1201 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     1261 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     1321 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     1381 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     1441 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     1501 ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta
     1561 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     1621 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     1681 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     1741 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     1801 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     1861 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     1921 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     1981 gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga
     2041 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2101 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2161 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2221 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2281 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2341 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2401 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2461 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2521 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2581 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2641 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2701 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     2761 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     2821 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     2881 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     2941 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3001 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3061 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3121 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3181 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3241 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3301 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3361 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     3421 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     3481 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     3541 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     3601 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     3661 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     3721 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     3781 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     3841 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     3901 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     3961 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4021 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4081 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4141 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4201 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4261 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4321 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4381 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4441 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4501 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4561 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4621 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4681 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     4741 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     4801 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4861 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4921 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     4981 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5041 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5101 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5161 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5221 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5281 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     5341 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     5401 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     5461 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     5521 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatccc
//
