LOCUS       sequence-450058-        7170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of ApyA peptide modified by the B12-rSAM enzyme ApyD and 
            the cytochrome P450 enzyme ApyO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7170)
  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 7170)
  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_ApyADO
FEATURES             Location/Qualifiers
     source          1..7170
                     /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             5261..5305
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(5343..5361)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5357..5404
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5634..6449)
                     /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(5634..6449)
                     /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     6357..6376
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6450..6541)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(6557..7170,1..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(6567..6584)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(6807..6826)
                     /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 taagattcaa ggagatatac
     3841 catgaacggt ctcccgcctt ctctgcctcg tgtggactca acagaaagcc tgttcgcgga
     3901 gcctttagca ttcttagcgc aagcccgttc tcgtcacgga gacgtgttcg ttatgcgcga
     3961 gcacggcccc atcttttccc gcgcatcgga ttgttcgggt gtcattgctg tctttggaga
     4021 gcaccgttta cgtcaaatcc ttacggacat cgacaacttc gcgttaccga tgagtgccgc
     4081 cgccaagatg gcattaccta agaacctcgt caatttaaac cgtggcctgc atagtatgcg
     4141 cgagccggag catggtcgtc acaagcgttt gcttaccggt acaattaacc gcgagctctt
     4201 tgacgcccac cgtttcgaaa ttcgcgcagc gctcaaccgc ttttgtgaaa tgctcaaggt
     4261 agatcgccgt atcagtgtgg tgagccgtat gcgcgagctg acggtcgaga tggcatctca
     4321 tatctttctg ggtgcacaat gtcaggaaga cgatgagttg gcgtttcttc tttcagccta
     4381 tttcaccctg cgccgcgagg ctagctcatt aaacgctcgt gacccgttgc tttatcgtga
     4441 cgagctgatc ggtgtgggac aacagctgga ccgcacatta cgcgaacgta tccgccgcta
     4501 tcgtaaacgc cccgtcgacg cccgcgccgg tctgttacag cgtcttgcta cggcaggtcc
     4561 gccgggctcg cctgcgttgt cggaagatga aattgtgggt cacgccaacg taatgttcgt
     4621 gtcttccacg gagccggttg ccatgtctct tacgtggttg ttgctggtct taagtcaact
     4681 gcctgactta cgtcgtgcgc tgcgtgcgga gatcgcggac cgtgcgtcta tgccggccag
     4741 tactaatggc gcgtcgtggt tagagaacgt tgtcaacgag acgttgcgct tattgacccc
     4801 aaatgccctg atggtccgcg ccacaacacg cgcagtgtca ctgcaaggtg ttgcactccc
     4861 ggcccgctgt gagattgtgg tatgtccatt tctggctcac cgcgaggcca aaccatttcc
     4921 cgacccccac gcattctcac catcgcgctg ggagaccgcg cgtcccagtc cttatgagta
     4981 ctttcctttc ggggcgggcg gacacttctg tgcggggcgt aatttggccc tctcacttat
     5041 tcgcgaagtg ttgtcgacgc tgttaagccg tttcgatttc gttcttgacg gtgagcaaag
     5101 catcgactgg cgcattcata ttatgctgat gcccaaggga gacccggctt tgattgctca
     5161 ccctgtggat gaacgtggcg ataccccgag tccaaagtgg cgtgggccca ttaccgattt
     5221 atttcacttc gcccctgggc tttcatgact cgagtctggt aaagaaaccg ctgctgcgaa
     5281 atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct aggctgctgc
     5341 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     5401 tttgctgaaa cctcaggcat ttgagaagca cacggtcaca ctgcttccgg tagtcaataa
     5461 accggtaaac cagcaataga cataagcggc tatttaacga ccctgccctg aaccgacgac
     5521 aagctgacga ccgggtctcc gcaagtggca cttttcgggg aaatgtgcgc ggaaccccta
     5581 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     5641 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     5701 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     5761 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     5821 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     5881 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     5941 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     6001 gcgagacgaa atacgcggtc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     6061 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     6121 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     6181 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     6241 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     6301 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     6361 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     6421 caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta ttgaagcatt
     6481 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6541 ataggcatgc agcgctcttc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga
     6601 ctgcggcgag cggtgtcagc tcactcaaaa gcggtaatac ggttatccac agaatcaggg
     6661 gataaagccg gaaagaacat gtgagcaaaa agcaaagcac cggaagaagc caacgccgca
     6721 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagcca
     6781 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6841 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6901 gggaagcgtg gcgctttctc atagctcacg ctgttggtat ctcagttcgg tgtaggtcgt
     6961 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     7021 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     7081 cattggtaac tgatttagag gactttgtct tgaagttatg cacctgttaa ggctaaactg
     7141 aaagaacaga ttttggtgag tgcggtcctc
//
