LOCUS       sequence-410398-        6566 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Tm9D8* TrpB for the synthesis of 7-Aza-L-Tryptophan 
            (7AW) from 7-Azaindole (7AI) and serine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6566)
  AUTHORS   Abdelkader EH, Qianzhu H, Huber T, Otting G
  TITLE     Genetic Encoding of 7-Aza-l-tryptophan: Isoelectronic Substitution 
            of a Single CH-Group in a Protein for a Nitrogen Atom for 
            Site-Selective Isotope Labeling.
  JOURNAL   ACS Sens. 2023 Oct 27. doi: 10.1021/acssensors.3c01904.
  PUBMED    37890165
REFERENCE   2  (bases 1 to 6566)
  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"; journalName: "ACS Sens. 
            2023 Oct 27. doi: 10.1021/acssensors.3c01904."
COMMENT     Sequence Label: pET-21(+)Tm9D8* TrpB
FEATURES             Location/Qualifiers
     source          1..6566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             3..25
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             36..53
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1242..1259
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1312..1330)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1326..1373
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1410..1865
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1497..1516)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1707..1728
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1891..1995
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1996..2064,2065..2856)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2214..2233)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3027..3615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3516..3535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3769..3786
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3801..3940
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3866..3885)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4026..4048
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4042..4233)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5042..6124)
                     /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     6086..6105
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6125..6202)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6408..6427
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6511..6530
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6511..6529
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6530..6554
                     /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)."
ORIGIN
        1 attttgttta actttaagaa ggagatatac atatgcacca ccaccatcac cataaaggct
       61 attttggtcc gtatggtggt cagtatgttc cggaaattct gatgggcgca ctggaagaac
      121 tggaagcagc atatgaaggc atcatgaaag atgagagctt ttggaaagaa tttaacgatc
      181 tgctgcgtga ttatgcaggt cgtccgacac cgctgtattt tgcacgtcgt ctgagtgaaa
      241 aatatggtgc acgtgtgtat ctgaaacgcg aggacctgct gcataccggt gcacataaaa
      301 tcaataatgc aattggtcag gttctgctgg caaagctgat gggtaaaacc cgtattattg
      361 cagaaaccgg tgcaggtcag catggtgttg caaccgcaac cgcagcagca ctgtttggta
      421 tggaatgtgt gatttatatg ggcgaagaag ataccattcg tcagaaactg aatgtggaac
      481 gtatgaaact gctgggtgca aaagttgttc cggttaaaag cggtagtcgt accctgaaag
      541 atgcaattga tgaagcactg cgcgattgga ttaccaatct gcaaaccacc tattacgttt
      601 ttggtagcgt tgttggtccg catccgtatc cgattattgt tcgtaatttt cagaaagtga
      661 tcggcgagga aaccaaaaag cagattccgg aaaaagaagg tcgtctgccg gattatattg
      721 ttgcatgtgt tagcggtggt agcaatgcag ccggtatctt ttatccgttt attgatagcg
      781 gtgtgaaact gattggtgtt gaagccggtg gtgaaggtct ggaaaccggt aaacatgcag
      841 caagcctgct gaaaggtaaa attggttatc tgcatggcag caaaaccttt gttctgcaag
      901 atgattgggg tcaagttcag gttagccata gcgttagcgc aggtctggat tatagtggcg
      961 ttggtcctga acatgcatat tggcgtgaaa ccggcaaagt tctgtatgat gcagttaccg
     1021 atgaagaggc actggatgca tttattgaac tgagccgtct ggaaggtatt attccggcac
     1081 tggaaagcag ccatgcactg gcatatctga aaaagattaa catcaaaggc aaggtcgttg
     1141 tggttaatct gagcggtcgt ggtgataaag atctggaaag cgttctgaat catccgtatg
     1201 ttcgtgaacg tattcgctaa aagcttgcgg ccgcactcga gcaccaccac caccaccact
     1261 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     1321 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     1381 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     1441 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1501 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1561 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1621 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1681 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1741 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     1801 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     1861 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1921 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1981 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2041 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2101 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2161 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2221 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     2281 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2341 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2401 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2461 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2521 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2581 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2641 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2701 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2761 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2821 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2881 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     2941 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3001 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3061 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3121 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3181 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3241 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3301 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3361 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3421 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3481 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3541 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3601 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3661 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3721 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3781 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3841 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3901 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3961 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4021 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4081 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4141 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4201 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4261 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     4321 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     4381 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     4441 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     4501 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     4561 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     4621 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     4681 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     4741 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     4801 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     4861 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     4921 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4981 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5041 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5101 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     5161 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     5221 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     5281 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     5341 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     5401 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     5461 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     5521 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     5581 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     5641 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     5701 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     5761 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     5821 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     5881 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     5941 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     6001 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     6061 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     6121 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     6181 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     6241 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     6301 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     6361 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     6421 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     6481 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
     6541 cggataacaa ttcccctcta ggatcc
//
