LOCUS       sequence-412884-        7352 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Precursor peptide CoiA1 and synthase CoiB, for the co-expression of 
            CoiA1 in E.coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7352)
  AUTHORS   Luo Y, Xu S, Frerk AM, van der Donk WA
  TITLE     Facile Method for Determining Lanthipeptide Stereochemistry.
  JOURNAL   Anal Chem. 2024 Jan 30;96(4):1767-1773. doi: 
            10.1021/acs.analchem.3c04958. Epub 2024 Jan 17.
  PUBMED    38232355
REFERENCE   2  (bases 1 to 7352)
  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/acs.analchem.3c04958"; journalName: "Anal Chem"; date: 
            "2024-01-30- 30"; volume: "96"; issue: "4"; pages: "1767-1773"
COMMENT     Sequence Label: pRSFDuet1_CoiA1_CoiB
FEATURES             Location/Qualifiers
     source          1..7352
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             131..424
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLKFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             431..451
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        698..716
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    717..741
                     /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             3889..3933
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3971..3989)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3985..4032
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4262..5077)
                     /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(4262..5077)
                     /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     4985..5004
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5078..5169)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5185..5934)
                     /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(5195..5212)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5435..5454)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6130..7212)
                     /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     7174..7193
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7213..7290)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(7336..7352,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgggttcttc
      121 tatggctaac atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga
      181 agtcaagccg gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt
      241 caagatcaaa aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg
      301 taaggaaatg gactccttaa aattcttgta cgacggtatt agaattcaag ctgatcagac
      361 ccctgaagat ttggacatgg aggataacga tattattgag gctcacagag aacagattgg
      421 tgggatcgag gaaaacctgt acttccaggg aatgaatgct aacaccatta aaggtcaggc
      481 acattcgcca gcagctaccg ccgggggtga tgcctttgac ctggatatct cagttttaga
      541 aagtgacgac ggctctgcga ccctgattaa cttgacggat gatggatgtg gctccacttg
      601 cagcagcccg tgcgcgacaa atgtggcgta atgcggccgc ataatgctta agtcgaacag
      661 aaagtaatcg tattgtacac ggccgcataa tcgaaattaa tacgactcac tataggggaa
      721 ttgtgagcgg ataacaattc cccatcttag tatattagtt aagtataaga aggagatata
      781 caatggggag ccgaaaagat tgccccttgt atcggtgcgc cgacactgcc ctggtaaggg
      841 ctgccagaag cgcgcgacta cccctgcctg catggcccga cctgacagat gatacaccgg
      901 attgtgaagt tcgctggcag acatggctgc gtgatgtttg gagtttaagt gaagcagccg
      961 actcaattga acaagcctca cccctattgg cacagcaagt gcaggcgcta tgctctgtcg
     1021 cgtcgcctga gacgcgtcag ctacgccgtg cggtagtctc cgtaatgcgg tacctgcttc
     1081 gcatgactgg acgggcgact ccgaatgggc tctttgccgg catcgctccc gcatcttttg
     1141 gcgaacgacc cggttggagc tggggtgaat ggcatcggcc ggttatacga gctgatggtg
     1201 attggatagc cgatttggtc gcacgattgg aggctgaccc tgagttactg agacaccttc
     1261 atgtaatggc caacaacacc attctcatca ggggggatcg gctcatcatg ccatacccac
     1321 cgcgttcctg tagaactgat gggtcccctg cagccgaagt atccgtgcgc tacacgagcg
     1381 ccgtgcggat cgcagtggat gccgctgctt cacccgtacc tgtccatgca gtagttgcgc
     1441 ttatgcgcag cgagtttcca gacgtgcccg ctgatcgcgt agaggggcta gtaagctctc
     1501 tggtcgagcg gggtgttctg atcagctctt tgcatgcacc gtcagcgaca ttggacgcgc
     1561 ttgatcacct tgtagaacag gctgaggctg cagcagcacc cagcgctacg gggctggtgg
     1621 cggatctgcg aacggttcgg gatgcaatcg cacgtcataa tcaggttctg gcacctgctg
     1681 atggtcggcg tctccgcacc gctctccgtc gcaaaatgac tgcgctctcg tcagttaagg
     1741 cccaaccgtt cacgctggat cttcgaatgg actgcagtct tactctgccg ccgcaagtcg
     1801 cgcgtgaagc tgagcaagca gctacagtac ttgcacgttt gtctgcatat ccgtttggta
     1861 cgccggcgtg gaaagatttt cataacagat ttttcgagcg ctatgggatc aactcacttg
     1921 tccctgtccg ggatttagtt gatccggatg tggggctggg cttcccagtt gggtatcgcg
     1981 atgcacaacc tgagcaagga gaagctctga ccgcccgcga acagcgcctg ctgtcgctcg
     2041 cccaggcagc agtgttagac ggacgtgacg agatccattt ggatgaagac cttatccgta
     2101 cgttaaccgt gggcgaccgc aatgcgccgc acgttccggc ccataccgaa ctccgctttc
     2161 gtattcgcgc cacgagccaa gaagctctcg aatcgggaga ttttgtcctt catggtattg
     2221 caccaagccg tggtatcggc acgactattg gtcgttcctt aggcttactg gcgcctgatg
     2281 agcaggcacg cgtggcggtt atgcttgagg agttaccagt caatactcct ggtgccgtac
     2341 ccacccaagt ctcatttact ccgttggacc gtggtgacac taacgtcact cgtgttactg
     2401 agctgttgcc agcagtaatc agtctggccg aacatcgccc agtggacgcc cgcacaattc
     2461 ctttagacga tctggcggtg ggctgcgatc gtcgtcgctt gtatttggtt agcctttcac
     2521 gtgcgtgcct gttggacccc atgactttgc atgcgcttga tttacgcgga catactcctc
     2581 ccttgggtcg tttcttaaca gagatttcac gcgcccagac ggctgtgtta acggtcttcc
     2641 cttgggccag tgctacggct ctgccgtatc tgccccgcgt tcgttacgga cgcgccattt
     2701 tgagccccgc gcgctggcgt ttagatcgct ctgaacttcc ggatcgtcgc gcttcctggg
     2761 gagagtggca taaagccgct gacgagtggc gcgcgcgccg ccgtgtgccg gatgaagtag
     2821 cagttgcaga aggtgaccag ttattaccct tagacctgtc ggagcgtgca cacttggcac
     2881 tgcttcgtgc ccacttagat acgcatgaga gtgtaatcct gactgaagcg gagtctgaag
     2941 aaggttggtt cgatggacgc tcccacgaga tcatcgcacc aatgacagct gtgcgccccc
     3001 cccaatggcc agccgtcccg ccggtcacgg cagatcagct ggttactcgc gaccacggct
     3061 atctgccagg tgcggcgtca tggttattgg ttaagttata cggacacgtt gatcgccaac
     3121 cagagatttt agccgaccac ttaccggcgc ttcttggtca gtgggacgaa ccccctacgt
     3181 ggtggtacat gcgctaccgt gatccacgtt ggcatttgcg tttgcgcatt gccgtgccaa
     3241 gtgagcaaga tttcgcattg accgcgcgtc gtgtctcttc ctgggccaac ggcttacgcc
     3301 gtgcgggatt attggcagat atgcaatttg caacttctta ccccgaaact ggtcgctggg
     3361 gcccaggccc cttaatgaac ttggcggaag atgtgttcgc agcagattcg cgcgcactgg
     3421 ccgttcaatt tgcccaatct agccgcccac accaacaagt attagccgcg gctaatttcg
     3481 ttagtgtggc cgctgcattc accggcagta cagccgcagg tatgaattgg ctgatcgcac
     3541 acggccgcat cactgatcct cgcccgatgg atcgcacggt ccgtgggcag gcgctgcgtc
     3601 tggcagaccc agcagatggt tgggctgcat tacgtgccgc ccccggtggc gacgctatcg
     3661 cgagtgcgtg gtcggagcgt gatgctgcct tggctcgtta ccgtcagaaa cttgacgcgt
     3721 tcggcggcat cgacttagat cttgtcttag actcattatt gcacgcccac cacattcgcg
     3781 ccgccggtat cgacaaggat gatgagcgta tgtgcgtacg tcttgctcat gccgccgcta
     3841 tggcttggac tcaccgcggt gatcatcatg agagtgccta agtctggtaa agaaaccgct
     3901 gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag
     3961 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     4021 aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact gcttccggta
     4081 gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc ctgccctgaa
     4141 ccgacgacaa gctgacgacc gggtctccgc aagtggcact tttcggggaa atgtgcgcgg
     4201 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     4261 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     4321 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     4381 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     4441 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     4501 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     4561 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     4621 gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg acaattacaa acaggaatcg
     4681 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     4741 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     4801 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     4861 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     4921 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     4981 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     5041 gcctagagca agacgtttcc cgttgaatat ggctcatact cttccttttt caatattatt
     5101 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5161 ataaacaaat aggcatgcag cgctcttccg cttcctcgct cactgactcg ctacgctcgg
     5221 tcgttcgact gcggcgagcg gtgtcagctc actcaaaagc ggtaatacgg ttatccacag
     5281 aatcagggga taaagccgga aagaacatgt gagcaaaaag caaagcaccg gaagaagcca
     5341 acgccgcagg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5401 tcaagccaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5461 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5521 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gttggtatct cagttcggtg
     5581 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5641 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5701 gcagcagcca ttggtaactg atttagagga ctttgtcttg aagttatgca cctgttaagg
     5761 ctaaactgaa agaacagatt ttggtgagtg cggtcctcca acccacttac cttggttcaa
     5821 agagttggta gctcagcgaa ccttgagaaa accaccgttg gtagcggtgg tttttcttta
     5881 tttatgagat gatgaatcaa tcggtctatc aagtcaacga acagctattc cgttactcta
     5941 gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca tacgatataa
     6001 gttgtaattc tcatgttagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     6061 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     6121 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6181 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     6241 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     6301 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     6361 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
     6421 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     6481 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     6541 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     6601 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     6661 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     6721 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     6781 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     6841 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     6901 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     6961 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     7021 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     7081 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     7141 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     7201 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     7261 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     7321 cctgcattag gaaattaata cgactcacta ta
//
