LOCUS       sequence-450059-        3783 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of unmodified ApyA peptide..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3783)
  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 3783)
  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_ApyA
FEATURES             Location/Qualifiers
     source          1..3783
                     /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             1874..1918
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1956..1974)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1970..2017
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2247..3062)
                     /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(2247..3062)
                     /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     2970..2989
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3063..3154)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(3170..3783,1..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(3180..3197)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3420..3439)
                     /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 actcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc
     1921 tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc
     1981 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaacctcagg catttgagaa
     2041 gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc
     2101 ggctatttaa cgaccctgcc ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg
     2161 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2221 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     2281 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     2341 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     2401 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     2461 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     2521 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     2581 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta
     2641 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     2701 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     2761 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     2821 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     2881 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     2941 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     3001 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     3061 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3121 tacatatttg aatgtattta gaaaaataaa caaataggca tgcagcgctc ttccgcttcc
     3181 tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggtgtc agctcactca
     3241 aaagcggtaa tacggttatc cacagaatca ggggataaag ccggaaagaa catgtgagca
     3301 aaaagcaaag caccggaaga agccaacgcc gcaggcgttt ttccataggc tccgcccccc
     3361 tgacgagcat cacaaaaatc gacgctcaag ccagaggtgg cgaaacccga caggactata
     3421 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3481 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3541 acgctgttgg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3601 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3661 ggtaagacac gacttatcgc cactggcagc agccattggt aactgattta gaggactttg
     3721 tcttgaagtt atgcacctgt taaggctaaa ctgaaagaac agattttggt gagtgcggtc
     3781 ctc
//
