LOCUS       sequence-331733-        7761 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-tagged BhaB7 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7761)
  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 7761)
  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-bhaB7
FEATURES             Location/Qualifiers
     source          1..7761
                     /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             2626..2643
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2696..2714)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2710..2757
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2794..3249
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2881..2900)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3091..3112
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3341..4156)
                     /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(3341..4156)
                     /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     4064..4083
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4148..4167
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4278..4866
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4767..4786
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5020..5037
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5052..5191
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5117..5136)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5277..5299
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5293..5484)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6293..7375)
                     /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     7337..7356
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7376..7453)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7659..7678
                     /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 atcattagca tttcaaatct ctcacctaag
      181 tctaatcgtt ctcagacaaa agattctcaa ataagcaaat ggagctattt ttccgattgc
      241 attttacgct caactggatt tcctgccgag tggatcgaaa agctgtgctt tcataaaacg
      301 acaacaatgt tcgatctatg ttatcgaacc gaacaatctt taaatgaatt ggttgagcgg
      361 ttaaaaacat tagaacaaac ttgtcatatc cctagtaatg cccgacaaga ttggcaaaga
      421 attcgcaagc tagtagggaa aagaaagaaa gttacagatg aattcattca tttaatggaa
      481 cagaaagggt ttgatcaatc cttaactcaa gaaatcgaaa gttatgaaag aaaacgtcgc
      541 cttcaagaaa aaaattggac aaaagctcgt gagtcatttg aggcagaatt agcggaaaag
      601 caattattcc tacaggagat ttataaaaat ccccgattgc aggaagccat ttttcaacaa
      661 agtccttcaa tgtataaaaa cgcagttgtc ccttatgttc actcttcttt acaaaaacgg
      721 aacacaaata ttaagcgaat tgaacgacag ttaatttcct atttacaaag actgtgtaca
      781 aaaaatgaaa ccacgagctt ctttggtcct attcagtatg gagtgttaac ctcagaacaa
      841 caagacattg aatataattt caatcagaag gaaactgaac ggagagcttt tatgccttat
      901 tggtcaatca aagtattagc agcacaaatg aaagaatgtg acgtattctt tccttatcta
      961 aatgtaaaac tttcctatca gtatacaaaa acaggtgatt caattcattc tcccatttta
     1021 gagaaaatgg tcaaatttcc aaagccatac gcggatatca tcaaacattt aacctatgaa
     1081 aatcaatcaa ttgagcagct cgcaaaaaaa ctttctcagc ctgtggcatg gttagcaaaa
     1141 aagcttcgac tattagaaaa aaagagattg atcctaattg agatccctct atcgatcaca
     1201 gagccggata gtttaaacga attaaggagt tggatactta aaattaaaga tgttgaacat
     1261 cccgacctat cactatggca agaaagattg gaatggcttt ggaaagtcaa acaagcttat
     1321 ccttacatga cgcttgggca gaaacaagag ctatttcaca aattggagtc aacttttact
     1381 gagtggacag gtgaacagcc taggcgttta ggcggggaga tttatgcaga tcgaaatctt
     1441 ttgtatgaag agtgtcatgg accgttaaac aatattaaaa ttggtgggtc cattaagcac
     1501 gttcttaaac aagatgtacc taagtggcta tcgatttgcg ctaaacacgg ggaacagagg
     1561 cggaaacaag aacaagcatt ggctcaagag atttttaaaa tgatgtatcc aaatgaagat
     1621 tctgtaccat ttctgaaatt cgttcacgac ctttctaatc acccagatgt acatacgtgg
     1681 gaaaagcgat ggcaatcaat aaaaacagaa atagaggagg ccattaccca tgaagtagcg
     1741 agtaatccta aatcagttgt ccatctttct ttagattacc aagactttga tcgtgacatg
     1801 gcatggttaa cttctcctga tctcatgata gctaaaaaag atgatgattc ctatcaagtc
     1861 attttaggcg aaatacatga cacgattatg gtttggggat gggccttaca atttcatcca
     1921 gaaaaagaca gagtgaatga acaattagta aagaaaatac aaaaaagtac gcaacacctt
     1981 cgaatgctta acatgctctc aagcaaacgc tttaaaattg taccttttga atatccaggt
     2041 acaacgatcc aaatgaacag cttttcaaac agcccaaatg aaaaaattcc gttaagtcaa
     2101 ttaaaagtaa cctacacgaa agaggggcta gcgcttacac ttcctgatca tgaagaggtt
     2161 ttttatacgt ataatgggga gttaaataca atggttcact cgtttttttc attgccccga
     2221 gccgtatctt tctcgatcag aacaggcgaa tcaactccac gtttgatgtt aggagacctt
     2281 gtcgttcaac gtgctaaatg gcacatttca aaacaggacg tctggagcaa aaaacacacg
     2341 ggctcttcaa tcgaattatt ttatgaggca taccgatttc acttaaaaga aagtctccct
     2401 aaagagggat ttgcaaaact tcctggccaa ccaaagcctg tctatgtaaa tttcactagt
     2461 tattttctta tagagatgtt ggagggactt tggccagacg gagagaattg tacgttcagc
     2521 gagatgttac caaacttgtc ggagctatgg ctttctaaag aagatcaaaa aactcacacc
     2581 gctgaaataa ggctgagtta ttttgttgaa aggagttaac tcgagcacca ccaccaccac
     2641 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     2701 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2761 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2821 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2881 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2941 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3001 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3061 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3121 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3181 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     3241 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3301 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     3361 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     3421 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     3481 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     3541 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     3601 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     3661 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     3721 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     3781 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     3841 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     3901 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     3961 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     4021 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     4081 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     4141 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     4201 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4261 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4321 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4381 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4441 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4501 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4561 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4621 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4681 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4741 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4801 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4861 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4921 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4981 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5041 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5101 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     5161 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5221 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5281 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     5341 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     5401 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     5461 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     5521 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     5581 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     5641 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     5701 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     5761 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     5821 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     5881 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     5941 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     6001 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     6061 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     6121 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     6181 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     6241 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     6301 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6361 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     6421 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     6481 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     6541 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     6601 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     6661 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     6721 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     6781 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     6841 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     6901 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     6961 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     7021 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     7081 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     7141 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     7201 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     7261 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     7321 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     7381 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     7441 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     7501 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     7561 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     7621 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     7681 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     7741 gagatctcga tcccgcgaaa t
//
