LOCUS       sequence-331737-        7791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-tagged AmmB3 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7791)
  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 7791)
  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: pET28a-ammB3
FEATURES             Location/Qualifiers
     source          1..7791
                     /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             2656..2673
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2726..2744)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2740..2787
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2824..3279
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2911..2930)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3121..3142
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3371..4186)
                     /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(3371..4186)
                     /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     4094..4113
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4178..4197
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4308..4896
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4797..4816
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5050..5067
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5082..5221
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5147..5166)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5307..5329
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5323..5514)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6323..7405)
                     /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     7367..7386
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7406..7483)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7689..7708
                     /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 atgacacgtg tttggacctt ggggacagtg
      181 tttgttgtgc gccatgccgg tatgccgttt gattggttag aaagtcttgg tggtagcgcg
      241 gatctccatg aggctgcaga gcgtttattg gatgctgaag aagaactccg cgcggcagcg
      301 ggcagtgcgt ttccgcgctt ggcggcggcg gtggaagaat gtgctcctga acgtttgccc
      361 ggtgtgccag caccggcacg tgaagcggct cgtgcgtggc gtgcggcggc ggcccgttat
      421 cgtgaagctc atcgtacggc cgatgaagca gcgacgaaag agttgcgccg tttgttagaa
      481 cgtccggaag tcgcggaagc ggtcttgtta agcaatccgg atgcgttacg caatatgtta
      541 cgtcctttac tggcgcatga tgggcccctc acgtcacgct atcgtcgcgc gcgtcgccaa
      601 ctttatacgt atgttcaacg cttttgtgcg aagaatgaaa cagtttcctt ctttggcccg
      661 atggcttatg gagaagtgga cccagggctt ccgactgcgg atggtgcact cttgagtact
      721 cgccttccgc gtacacgccg tgtatttgtg agccattggg caggtcgtgc gctcttgcgc
      781 gctgttacgc gtgatacagc tttgttaccg gatttaccgt ttcatcgtac gcgtgatggt
      841 gagccggcgt ggcctgatgg agccagcccg gaagatgcgg ccgcggcacg tacagttttg
      901 accgcactga cgccggatgg tacgactctt cgtggtctgg tgcgtatttt aggtacgccg
      961 gcacgtgaac tcgctcgtgc cctgcgtctc ttggtggcag cagatgctgc tgatgtgggt
     1021 cttggtggtg gaccgtttga tctggagcct ctgacgactt tgcgtgatgg tctggatcgc
     1081 cttccccctt cggcagcacg tgatcgctgg attgctcgta ttgaaggtct tgaagcgttg
     1141 cgcgccgctc tgcaagctgc tggaccggat gagcgcgaag cggctattgg cgcgttagaa
     1201 cgcgcgttta cggaagcgac gggtgagccg gcacgtcgtg cagcggggtc gacgtatgct
     1261 gatcgtgcag tgttttatga agaaggtagc tcgccgtttg cactccgcat tggtcctgcc
     1321 ttagcggcgc gttgggaaca acgcttacgt ggtgtgttgg aactgtgtac cgcgcatggt
     1381 catcttactc aacgcgcggc ggcagatgcg gtccgtagcg cgctgggtac gggcgcgcgc
     1441 ttagatttgg aacgctatgc cgcggcggct gcagaagctt ttccggcgcc gggttctgca
     1501 tttgcagtgg cacatgcccc ggattatcct gccgccgatc gtgagcgcca tacaactcgt
     1561 ctccgtgaat tagcggatgg tcttcgtggt gatcgttatg cattggtgga tttgtgtcct
     1621 cgcgcttctg gcgcggcggg tctggctgat gcagatctgg tggtggcgcg tgttcatcat
     1681 cacttacttg tgccgggttg gctggatacg atgcatccgg agccgcgtcg ttttgctgcc
     1741 gcagcagcag attgggtagc gcgtcaagat ggacgcgtgg ttggttttga ttttggtcgt
     1801 cgtaataaag gttattatcg ctttcctggt cgtgcagtag ctctgcgtcc ggcgtcatgg
     1861 actgatgcgg gtgatccacc tcttcgtccg cgtgatctga ctgtggaact tggtgaaccc
     1921 cctggtcctg ccgcaggctc gggtcatggt tatgaaacgg gtccgggtta tgaaccgggc
     1981 tatggccgcg gcagcgcaag cggatcaggt actggcgtac gccttctcga cccagatggt
     2041 cgtgaagtga cggcatatat tccgttgaat gattttgtta aatatgcacc atttgccgct
     2101 ctctcacatc ctcaagtact gcatcccgcc tttacagcgc cggaagctgc gccagaggca
     2161 gcgccgcaag tagcctctga tgcagcgcgt gatgcagctc cagatgatcc acctggtggt
     2221 gcgcctggta ccgcacatgg tggtaccgcc ccggtagatg ctgcgcatgg tggaaccgat
     2281 ccaggggcac ttccagaagt ggctgttgat ggtgtggttt tacaacgtcc gcgttggcgt
     2341 gttgatgcga gccgtttacg tcgcagcgct cctcatgcac gtttcttaga gttacgtcgt
     2401 ttggcgcgtc gtacgggttt acgttttctg ttttgtcgtg caccgcgtga acgtaaaccc
     2461 tatctgttgg atttagcctc accgttagcg gctgatcttg cggctcacat tggacatcgt
     2521 gaagatgtac tcacggctga agccatgtca ccagccccgg gtgatttgtg gttgcgtgat
     2581 gaacaaggtc gtcgttatac atgcgaattg cgtatgcaat ttacgggtcg tgaagtggcg
     2641 gcgccctaac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     2701 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     2761 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     2821 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2881 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2941 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3001 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3061 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3121 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3181 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3241 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     3301 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3361 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     3421 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     3481 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     3541 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     3601 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     3661 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     3721 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     3781 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     3841 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     3901 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     3961 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     4021 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     4081 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     4141 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     4201 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     4261 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4321 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4381 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4441 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4501 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4561 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4621 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4681 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4741 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4801 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4861 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4921 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4981 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5041 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     5101 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     5161 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     5221 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5281 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5341 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     5401 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     5461 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     5521 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     5581 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     5641 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     5701 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     5761 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     5821 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     5881 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     5941 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     6001 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     6061 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     6121 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     6181 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     6241 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     6301 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6361 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     6421 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     6481 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     6541 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     6601 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     6661 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     6721 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     6781 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     6841 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     6901 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     6961 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     7021 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     7081 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     7141 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     7201 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     7261 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     7321 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     7381 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     7441 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     7501 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     7561 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     7621 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     7681 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     7741 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t
//
