LOCUS       sequence-331735-        7137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses His6-tagged AmmC1 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7137)
  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 7137)
  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-ammC1
FEATURES             Location/Qualifiers
     source          1..7137
                     /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             2002..2019
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2072..2090)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2086..2133
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2170..2625
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2257..2276)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2467..2488
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2717..3532)
                     /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(2717..3532)
                     /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     3440..3459
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3524..3543
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3654..4242
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4143..4162
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4396..4413
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4428..4567
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4493..4512)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4653..4675
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4669..4860)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5669..6751)
                     /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     6713..6732
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6752..6829)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7035..7054
                     /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 atggtcaatc gtcccagtga tccgcgtatc
      181 tcttcactcc tcgatggggc acgtactccg ttaccaggcg gcgctggtac ggtgcgtgtg
      241 cctgttgata cgggtcctgc tgctagctat gccggtttgc gtgcggcact ggctcgctta
      301 cgtgcagaag gagccgattt tggtggcctt atctcacgtc atgggccgga atgggcggaa
      361 ttgtttcccg cagatgctcc tggtgaaccc ccactggcgg aaacggcatt agctccatcg
      421 gaacgtcgtc ttcatcgtga atccgaacaa aattatcgtg ttctggcggt ggcagctagc
      481 gccttatgta ccggtgcgga acaaacgggg cgtccattag aacttacggg cgtaggcggc
      541 actgatctcg cctcgcttcg tgggtttatg cgcgcacatg aatttgcacg tacgcgtcca
      601 ggcgtacgca ttcttttagc ggatcctact gctgttcgtg ctgcggtgct cccggaagcg
      661 gattatcgcg cggagcgtgc gctctgtttg cgtcgcatgg gtgttggggc agctcctgcg
      721 gatttacgtc cggtattacc gacccgtccg gaagcaggtc caggccttgg cgatggtggt
      781 acggctgatg ctcgcttgta tgcagacgca tttggtgatg gtacagcgtt tgatcgtttg
      841 gcagcggtgt tagcggggtg tcgtcgtggg ttctttacgg gtaattggga agcaatggcg
      901 gcgctggcag cgactggtgg tactctcctt gatgggtttc cggatagtcg cgtggcagat
      961 ttattggctg cagcgcgtga gcaagatcaa caagcggaag ctattgaatt tgaaccgggc
     1021 attctccgta ctacggatga tgttcgcgcg tttctggcga aagtgctggg cgtccaagcg
     1081 acgtttcgtg gtgatcaaga tcgcgccctt gcacattttc gtgcaatgcg tgccggcgaa
     1141 cgtttaagtc ctgaagtacg tgcgcaatcg catttatatg cggctctgac attgactaag
     1201 cgcctgggtt cagtggatga agctgttgcg gaagtcgaag aaggttttac ggcagtgcct
     1261 catcgtgaag gagaaggcaa tggggtacgc cgtgaacgtg gatggctcca taatttgcgc
     1321 gctttaacgc attttgcacg tcgcgaactc cgtgaagctt ttggtcatga gaaacaagcg
     1381 ttagcttgta ttgaaggtct ggatgatccg agctcaattc acttgcgcgt aaatctgttt
     1441 agcaatgtat ctgtcctgca agaaaaggcc gggcgtcatc aacaagcagc gcgtacgtgg
     1501 tcgcgtttta aagaagcaga aggtagcgcg aatgccacct ttgtgaaaca tcatgcttat
     1561 cgcgcaggtg gcttagcgtt gctctgtggg gatcgtgata ctgcccgtga agatcttgcg
     1621 aaaagcattg cgtgtgccgc cgaatttaat gatgattttc atgaagcgga aattcgcacc
     1681 gaagtcggca cgcttttact ggcggatgga gatcgtgcag gagcggcggc ccattttgaa
     1741 cgcgctgttg cgctgactag ccgtattggg gatccgtatc gtatggcact ggcacaactg
     1801 ggacgtgcgc gtgcgggtgg tgcggatcct gatccgtcag tggctgcatt agctcgtctt
     1861 tcaacgactc atccggcgcg tggagccgca cttgcggatg cggcagagcg tggtggtgat
     1921 gccgcagatg ggttgcttcc cgcagttcgt acaaaactga atcgtccttt tgatctcatt
     1981 aattttcaag aataactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     2041 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     2101 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     2161 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2221 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2281 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2341 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2401 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2461 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2521 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2581 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     2641 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2701 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2761 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2821 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     2881 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2941 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     3001 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     3061 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     3121 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     3181 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     3241 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3301 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3361 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3421 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3481 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3541 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     3601 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3661 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3721 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3781 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3841 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3901 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3961 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4021 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4081 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4141 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4201 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4261 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4321 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4381 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4441 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4501 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4561 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4621 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4681 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4741 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4801 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4861 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4921 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4981 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     5041 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     5101 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     5161 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5221 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5281 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5341 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5401 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5461 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5521 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5581 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5641 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5701 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5761 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5821 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     5881 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     5941 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     6001 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6061 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6121 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6181 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6241 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6301 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6361 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6421 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6481 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6541 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6601 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6661 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6721 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6781 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     6841 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     6901 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     6961 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     7021 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     7081 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
