LOCUS       sequence-450052-        5745 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of ApyA peptide modified by the B12-rSAM enzyme ApyD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5745)
  AUTHORS   Nguyen DT, Zhu L, Gray DL, Woods TJ, Padhi C, Flatt KM, Mitchell DA,
            van der Donk WA
  TITLE     Biosynthesis of Macrocyclic Peptides with C-Terminal 
            beta-Amino-alpha-keto Acid Groups by Three Different Metalloenzymes.
  JOURNAL   ACS Cent Sci. 2024 Apr 11;10(5):1022-1032. doi: 
            10.1021/acscentsci.4c00088. eCollection 2024 May 22.
  PUBMED    38799663
REFERENCE   2  (bases 1 to 5745)
  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: 
            "10.1021/acscentsci.4c00088"; journalName: "ACS Cent Sci"; date: 
            "2024-04-11- 11"; volume: "10"; issue: "5"; pages: "1022-1032"
COMMENT     Sequence Label: pRSF_ApyAD
FEATURES             Location/Qualifiers
     source          1..5745
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(332..1414)
                     /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     1376..1395
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1415..1492)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1538..1557
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1538..1556
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1557..1581
                     /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)."
     CDS             1637..1654
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3836..3880
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3918..3936)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3932..3979
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4209..5024)
                     /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(4209..5024)
                     /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     4932..4951
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5025..5116)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5132..5745,1..136))
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(5142..5159)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5382..5401)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 caacccactt accttggttc aaagagttgg tagctcagcg aaccttgaga aaaccaccgt
       61 tggtagcggt ggtttttctt tatttatgag atgatgaatc aatcggtcta tcaagtcaac
      121 gaacagctat tccgttactc tagatttcag tgcaatttat ctcttcaaat gtagcacctg
      181 aagtcagccc catacgatat aagttgtaat tctcatgtta gtcatgcccc gcgcccaccg
      241 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
      301 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
      361 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
      421 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
      481 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
      541 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
      601 ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
      661 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
      721 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
      781 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
      841 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
      901 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
      961 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     1021 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     1081 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     1141 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     1201 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     1261 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     1321 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     1381 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     1441 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     1501 cgacgctctc ccttatgcga ctcctgcatt aggaaattaa tacgactcac tataggggaa
     1561 ttgtgagcgg ataacaattc ccctgtagaa ataattttgt ttaactttaa taaggagata
     1621 taccatgggc agcagccatc accatcatca ccacagccag atggcgacca aaccaaaaaa
     1681 gaccaagggc gtgagcgtat ctattgaagg gaaattaccg aaaatgacgt tagatatgcc
     1741 tgtcgatgca aaaaaaatca aagctattca gaaatgtctg gagaacggta aactcaccat
     1801 taccatgtct aaagtagatc tggcaggcgg tcgtatgggg gaaggttatc tgtacgattg
     1861 agattcaagg agatatacca tgaaactgtt gctggttgat aacttggtaa tgccggagga
     1921 gggctcgctg gcatatttgg acgtgcatcc gcatctggga ctgctggcgc tggcggccgt
     1981 cgcagaagca gacggtcacc gcgtgcaaat ttacgatccg aaacgcctga ttcgtgatgg
     2041 cacactggct tatgatgcca ccctttatga acgcgcatct caagcgattc tggcagcgcg
     2101 tcctgatggc gtcggtttca ctgcactggg ctgttccttc ctgttcgcgt tgaatgtggc
     2161 ggcgttactg aaacgtcgcg aacccgatct gccggtgttg ctgggcggtc cgcacgcgac
     2221 catgctgcat cgccagattc tcgaacgttt tccgcagttt gatatcatcg ttcgctatga
     2281 agccgatgaa attctgccgg ccgtcctcga ttgcctgcca caccgcacct tcgacgtgat
     2341 ccctggcctg agttggcgtg caaccggtcg cggcagcccg cttcggttca cggatggcaa
     2401 gccgaaagtt gaggatctgg acctgctgcc catcgctagt tacgaccatt atccggttga
     2461 agaactggga ctgtcgatgc tgcgtatcga agccggccgc ggctgcccat ttgcgtgcac
     2521 cttttgttcc acggccggtt tctttcagcg ttccttccgc ctgaagtccg ctgaacggct
     2581 ggtgcgtgaa cttgatatct tacaccaacg ttaccgtgtc tccgatttca aactggatca
     2641 tgatatgttt actgtgaatc gtcgtaaagt catggaattc tgtgaagcgg ttgcaggtcg
     2701 tgattatcgc tggcgtgcct cggcccgcat cgactgtgtg gatgaagccc tgctgaagaa
     2761 aatggcggat gcgggttgcg ttaacctgta ctttggtgtt gaaacaggtt cagaacgcat
     2821 gcagaaactg tgtaaaaagc gtctggatct tcaacgtgtg gagccaatcc tggcggccgc
     2881 ggacagcttc ggcattgaaa ccaccgcctc gttcatcacc ggctacccgg aagaaacggg
     2941 tgaagatcaa gacgatacgc tcgatatgat tggccgctgc gcccgtcgtg ctagttgtct
     3001 gacccagctg catatgctgg cgccggaacc ggggacgccg ttgttcgacg aacgcggcgc
     3061 ggaaatcgcg tatgacggtt gtggcggtcc ctataacacg cgtttattat ctagctcaga
     3121 tgaacgtgcg gtactcggtc accctgatat tttccagact tactaccact atccggcagc
     3181 tttaccgcgt gcacgttata tctttgctgt tggggctgcc gatctcctgc gtcgtgtggg
     3241 tccagtaatt tttgcgtatg cgctgcgtgg attcggcggc cgtctgtcca ctctgattag
     3301 tgcgtggcgc cagttcgccg acgagaccag tccgggagcc ccggcggacg cagcgggtct
     3361 ggaagcgttt atcgctgctc gttttggccg gggccaccac ctgcatagtc tgttccgtta
     3421 tgcgttgcgc ctgtacgccg cccgtgcccc tgcagatgtg atcgtcgaag gtccatatga
     3481 gccagatcgt ccatgcatgc ttgctgaaaa cgtcgggttg ttggaagaca ttcacaattg
     3541 tgatgccttg gttgatcgta ttcgccgttc aaccgaaggc gccccgatgc tgggcgatgc
     3601 cgatgtcggg ggcttgggca cgtatatcgt gcagggtcac ggcgaagccg ccacaggtta
     3661 ctgggtcgaa tccggtattg cgactctgct ggatctgttt cgttctcctc gtagttgccg
     3721 ggctgtggcg caatggctgg cagacgcaac gcgtaacgat ggcattgatg ccagcatctt
     3781 tgaacctctg ctccgccgcg gtattctcgt gagcgcaggg taactcgagt ctggtaaaga
     3841 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     3901 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     3961 ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct
     4021 tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg
     4081 ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt cggggaaatg
     4141 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4201 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     4261 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     4321 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     4381 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     4441 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     4501 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     4561 cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca attacaaaca
     4621 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     4681 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     4741 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     4801 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     4861 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     4921 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     4981 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt cctttttcaa
     5041 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5101 tagaaaaata aacaaatagg catgcagcgc tcttccgctt cctcgctcac tgactcgcta
     5161 cgctcggtcg ttcgactgcg gcgagcggtg tcagctcact caaaagcggt aatacggtta
     5221 tccacagaat caggggataa agccggaaag aacatgtgag caaaaagcaa agcaccggaa
     5281 gaagccaacg ccgcaggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5341 tcgacgctca agccagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5401 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5461 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgtt ggtatctcag
     5521 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5581 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5641 gccactggca gcagccattg gtaactgatt tagaggactt tgtcttgaag ttatgcacct
     5701 gttaaggcta aactgaaaga acagattttg gtgagtgcgg tcctc
//
