LOCUS       sequence-331729-        7485 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-tagged BhaB1 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7485)
  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 7485)
  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-bhaB1
FEATURES             Location/Qualifiers
     source          1..7485
                     /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             2350..2367
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2420..2438)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2434..2481
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2518..2973
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2605..2624)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2815..2836
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3065..3880)
                     /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(3065..3880)
                     /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     3788..3807
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3872..3891
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4002..4590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4491..4510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4744..4761
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4776..4915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4841..4860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5001..5023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5017..5208)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6017..7099)
                     /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     7061..7080
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7100..7177)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7383..7402
                     /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 tggttcaata cttccacatg gcgttggtta
      181 aagccatttg tgctccgcag tacctctatc cccatcgaga tggttttgaa cttaaggtta
      241 aaagaatcaa taaagcaact aaatgaatgg agcaaaagcc aacaatcatt tgatcaatgg
      301 tgcaaccatt ggattaaaga aattaggtct gtaccagtag agcgaataag tcttccgcac
      361 caacgaaaag tcatacgaag aataaaaaaa agaaacccac taaccgaaca agataaaatc
      421 attgttacca agtggggaat ggaaaagttg attgatgagt actgctcaca gcagcaatca
      481 ttaataagct taaagaaaaa ggcgcaacaa tcttttcatg aagagtttga acgactacaa
      541 caacagttaa ttgagaggtt tcaatctagc caatatgaat tagctctgta tacattgaac
      601 cacaagcttt ggcatttctg gattaaggag aatcgtcttg aaaatctaac tgatgcacaa
      661 aaaaaacaaa catgtagaac ccttttcgcc tatcttcaac gggtctcaac taaaaatgac
      721 actatcggtg aatatggccc tatcagttat ggtgcattca atcaaggcaa gctgattcaa
      781 actaggcaaa ttacgaaaag aaaagcctat tttgcctatc aagggctaca aaaattgcta
      841 agtgttataa aaaaggattt aatgccgcag gctcatgcct ggaatctccc atcgtccacg
      901 gtagatgggt ggaagcttgt tagagaacgg gttgaagatt gtaatgatcc tcgttcagaa
      961 atatggatac gaatcctaga tcaacttgag gagactagac atttgtttga gacgtccact
     1021 caaccaaacg aaaaagaaca agccttagct caagctgaga aactatttac aaacataaca
     1081 ggggaggctt ttcaaggaga gaatcagcaa tacttcgcag ataaaacatt gctgtatgaa
     1141 gaatgccatg atcagcaata tttacccgtc accctatctg tcgatttatg gacagagtta
     1201 gatcatgctt tgttgattca tgccaaacta cgcctagaca tttggagatc ttggcaaaat
     1261 attgcggtcc agaaattcga gcaattggcg aatggaaatg tttcgattcc gttaatgaaa
     1321 tgggtttcct attggttgcg ttatcctcca agccttgaca accaatcagc aaccgatctg
     1381 ctttcacctt ggctagttga tcgtaatggc tacctttcaa ttaaactccc ggaccgagtt
     1441 atccgaagct ggcggaagga gattaactca ttcagatggt tactctcacc tgatttaatg
     1501 gtaaaaggaa aatttgagac taatcgtttc aatgtagcat ccatcgtgat aggggaatta
     1561 catcacggtt ttaccgccga tggttggatg tttgaatttc atcctgacaa aaaagaaatc
     1621 aatcgaatag tccggcaagc tatgccagaa aagaatccaa gctttacttg ggctaattgg
     1681 atctttcaaa ggaaaatgaa aagtactccg caagagtacc caggtgtatc agtaaagctt
     1741 acaggtcatt ctgaataccc agatgagaag tcattctctc tatatgactt ggaggttaga
     1801 aggttaggcg ataaagtggc tgttttttta caaggaactg ataatgctct aatgttttat
     1861 ccccctgcct atggatttaa cgaaacaagc tttttccctt ttgcactttt ctgttcacct
     1921 atgatggaac cttcatccaa ttactcaaaa gggagacaca aagcaataaa gttaggaaat
     1981 gtcacattaa ttagggaaca ttgggttttc gagccgacag attggggaga agtccatgca
     2041 tcacttgatg gatttcaaca aatggcgtgt ttacaagaga ttaaggttaa atatcaatta
     2101 ccgcaagtag gatacttaag attttctcaa gagctaaaac ctgtttggtt tgactttaat
     2161 aatccatttt gtgtcgattt gtttttcaat atggctaaaa aagcagaatg gtttacgttt
     2221 tccagaatgg acccaggggt ggaggattta tttctgaagg atgatcgagg ccattattgt
     2281 tgtgaattgc gaacctttgc ttatcggcaa gagataactt ccaaagatat gctcatgggt
     2341 taactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     2401 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     2461 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     2521 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2581 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2641 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2701 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2761 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     2821 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     2881 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     2941 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     3001 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     3061 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     3121 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     3181 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     3241 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     3301 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     3361 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     3421 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     3481 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     3541 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     3601 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     3661 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     3721 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     3781 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     3841 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     3901 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     3961 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4021 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4081 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4141 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4201 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4261 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4321 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4381 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4441 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4501 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4561 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4621 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4681 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4741 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     4801 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     4861 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     4921 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     4981 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5041 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     5101 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     5161 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     5221 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     5281 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     5341 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     5401 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     5461 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     5521 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     5581 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     5641 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     5701 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     5761 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     5821 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     5881 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     5941 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     6001 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6061 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     6121 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     6181 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     6241 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     6301 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     6361 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     6421 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     6481 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     6541 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     6601 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     6661 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     6721 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6781 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     6841 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     6901 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6961 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     7021 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     7081 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     7141 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     7201 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     7261 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     7321 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     7381 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     7441 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaat
//
