LOCUS       sequence-450053-        5208 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of ApyA peptide modified by the cytochrome P450 enzyme 
            ApyO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5208)
  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 5208)
  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_ApyAO
FEATURES             Location/Qualifiers
     source          1..5208
                     /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             3299..3343
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3381..3399)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3395..3442
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3672..4487)
                     /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(3672..4487)
                     /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     4395..4414
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4488..4579)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(4595..5208,1..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(4605..4622)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4845..4864)
                     /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 tgaacggtct cccgccttct ctgcctcgtg tggactcaac
     1921 agaaagcctg ttcgcggagc ctttagcatt cttagcgcaa gcccgttctc gtcacggaga
     1981 cgtgttcgtt atgcgcgagc acggccccat cttttcccgc gcatcggatt gttcgggtgt
     2041 cattgctgtc tttggagagc accgtttacg tcaaatcctt acggacatcg acaacttcgc
     2101 gttaccgatg agtgccgccg ccaagatggc attacctaag aacctcgtca atttaaaccg
     2161 tggcctgcat agtatgcgcg agccggagca tggtcgtcac aagcgtttgc ttaccggtac
     2221 aattaaccgc gagctctttg acgcccaccg tttcgaaatt cgcgcagcgc tcaaccgctt
     2281 ttgtgaaatg ctcaaggtag atcgccgtat cagtgtggtg agccgtatgc gcgagctgac
     2341 ggtcgagatg gcatctcata tctttctggg tgcacaatgt caggaagacg atgagttggc
     2401 gtttcttctt tcagcctatt tcaccctgcg ccgcgaggct agctcattaa acgctcgtga
     2461 cccgttgctt tatcgtgacg agctgatcgg tgtgggacaa cagctggacc gcacattacg
     2521 cgaacgtatc cgccgctatc gtaaacgccc cgtcgacgcc cgcgccggtc tgttacagcg
     2581 tcttgctacg gcaggtccgc cgggctcgcc tgcgttgtcg gaagatgaaa ttgtgggtca
     2641 cgccaacgta atgttcgtgt cttccacgga gccggttgcc atgtctctta cgtggttgtt
     2701 gctggtctta agtcaactgc ctgacttacg tcgtgcgctg cgtgcggaga tcgcggaccg
     2761 tgcgtctatg ccggccagta ctaatggcgc gtcgtggtta gagaacgttg tcaacgagac
     2821 gttgcgctta ttgaccccaa atgccctgat ggtccgcgcc acaacacgcg cagtgtcact
     2881 gcaaggtgtt gcactcccgg cccgctgtga gattgtggta tgtccatttc tggctcaccg
     2941 cgaggccaaa ccatttcccg acccccacgc attctcacca tcgcgctggg agaccgcgcg
     3001 tcccagtcct tatgagtact ttcctttcgg ggcgggcgga cacttctgtg cggggcgtaa
     3061 tttggccctc tcacttattc gcgaagtgtt gtcgacgctg ttaagccgtt tcgatttcgt
     3121 tcttgacggt gagcaaagca tcgactggcg cattcatatt atgctgatgc ccaagggaga
     3181 cccggctttg attgctcacc ctgtggatga acgtggcgat accccgagtc caaagtggcg
     3241 tgggcccatt accgatttat ttcacttcgc ccctgggctt tcatgactcg agtctggtaa
     3301 agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta
     3361 attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     3421 acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact
     3481 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
     3541 ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc aagtggcact tttcggggaa
     3601 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3661 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     3721 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     3781 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     3841 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     3901 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     3961 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     4021 attcgtgatt gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg acaattacaa
     4081 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     4141 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     4201 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     4261 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     4321 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     4381 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     4441 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcatact cttccttttt
     4501 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4561 atttagaaaa ataaacaaat aggcatgcag cgctcttccg cttcctcgct cactgactcg
     4621 ctacgctcgg tcgttcgact gcggcgagcg gtgtcagctc actcaaaagc ggtaatacgg
     4681 ttatccacag aatcagggga taaagccgga aagaacatgt gagcaaaaag caaagcaccg
     4741 gaagaagcca acgccgcagg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4801 aaatcgacgc tcaagccaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4861 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4921 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gttggtatct
     4981 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5041 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5101 atcgccactg gcagcagcca ttggtaactg atttagagga ctttgtcttg aagttatgca
     5161 cctgttaagg ctaaactgaa agaacagatt ttggtgagtg cggtcctc
//
