LOCUS       sequence-334446-        5539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-tagged BhaA in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5539)
  AUTHORS   Daniels PN, Lee H, Splain RA, Ting CP, Zhu L, Zhao X, Moore BS, van 
            der Donk WA
  TITLE     A biosynthetic pathway to aromatic amines that uses glycyl-tRNA as 
            nitrogen donor.
  JOURNAL   Nat Chem. 2022 Jan;14(1):71-77. doi: 10.1038/s41557-021-00802-2. 
            Epub 2021 Nov 1.
  PUBMED    34725492
REFERENCE   2  (bases 1 to 5539)
  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"; doi: 
            "10.1038/s41557-021-00802-2"; journalName: "Nat Chem"; date: 
            "2022-01"; volume: "14"; issue: "1"; pages: "71-77"
COMMENT     Sequence Label: pET28b-bhaA
FEATURES             Location/Qualifiers
     source          1..5539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             404..421
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(474..492)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      488..535
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      572..1027
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(659..678)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     869..890
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1119..1934)
                     /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(1119..1934)
                     /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     1842..1861
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2056..2644
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2545..2564
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2798..2815
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2830..2969
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2895..2914)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3055..3077
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3071..3262)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4071..5153)
                     /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     5115..5134
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5154..5231)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5437..5456
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gccgacaagg ttacacctga agaagagtta
      181 gatctagaat tagaaattga agaccttgat gacatcgact ttgatctaga ggaaattgaa
      241 gataaagtcg ctcctctggc tctttaattc gggaaacctt ctgtatagga atctgctaag
      301 tgtgagcaga ttcctataca caataagcgt ttataagggg aacttgaaat gcaaaacttg
      361 catatttttg gagaacaaaa aaaaacagac tgggtgactc gagcaccacc accaccacca
      421 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
      481 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
      541 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
      601 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
      661 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
      721 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
      781 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
      841 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
      901 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
      961 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     1021 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1081 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     1141 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     1201 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     1261 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     1321 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     1381 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     1441 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     1501 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     1561 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     1621 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     1681 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     1741 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     1801 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     1861 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     1921 tgaatatggc tcataactcc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     1981 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2041 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2101 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2161 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2221 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2281 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2341 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2401 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2461 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2521 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2581 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2641 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2701 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2761 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2821 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     2881 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     2941 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     3001 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     3061 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     3121 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     3181 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     3241 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg
     3301 taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg
     3361 cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga
     3421 gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg
     3481 gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc
     3541 gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg
     3601 cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct
     3661 aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc
     3721 gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     3781 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     3841 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     3901 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     3961 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4021 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4081 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4141 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     4201 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     4261 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     4321 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     4381 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     4441 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     4501 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     4561 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     4621 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     4681 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     4741 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     4801 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     4861 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     4921 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     4981 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     5041 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     5101 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     5161 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     5221 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     5281 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     5341 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     5401 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     5461 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     5521 gatctcgatc ccgcgaaat
//
