LOCUS       sequence-220763-        6178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of hexahistidine-tagged AceS methylase in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6178)
  AUTHORS   Sheehan J, Murphy CD, Caffrey P
  TITLE     New insights into polyene macrolide biosynthesis in Couchioplanes 
            caeruleus.
  JOURNAL   Mol Biosyst. 2017 May 2;13(5):866-873. doi: 10.1039/c7mb00112f.
  PUBMED    28383583
REFERENCE   2  (bases 1 to 6178)
  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"; journalName: 
            "Mol Biosyst"; date: "2017-05-2- 2"; volume: "13"; issue: "5"; 
            pages: "866-873"
COMMENT     Sequence Label: pET28-MetN
FEATURES             Location/Qualifiers
     source          1..6178
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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)."
     RBS             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             335..352
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1251..1268
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1321..1339)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1335..1382
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1419..1874
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1506..1525)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1716..1737
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1966..2781)
                     /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(1966..2781)
                     /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     2689..2708
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2773..2792
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2903..3491
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3392..3411
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3645..3662
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3677..3816
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3742..3761)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3902..3924
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3918..4109)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4918..6000)
                     /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     5962..5981
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6001..6078)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatgac
      361 catcggcaca ctcatcgacg cggcggtcgg tccgatagcg gagggcatga gccccacccc
      421 gcaccgcgac gccgaggagc tgtacgcgct tgcggccggc atcgacgtcg cggacatcga
      481 tcgcgacggc gagccggacg agcagaccac cgacgagttc cagcgcctca cccgcagccg
      541 cgtctccgga gtgccgatgg cctatctgac cggtgtggcc ccgttcgccg ggctggagct
      601 acgggtcggc gcaggcgtct tcgtgccccg gcgtgacttc agcgacgttg ttgagcccgt
      661 cctgtcccac ctcaccggcg tcaccgcgcc cgtggtcgtc gacctgtgcg cgggcgccgg
      721 ggcgcacgcc ctggtgatcg cccaccgccg ccccgacgcc gaggtgcacg cggtggagat
      781 cgacgaggag gcgtacggct tcgccgagct caacgccgcc gagcgcgtgg ccgggggcga
      841 caccggcatc cacctgcacc ggggcgatgt cacgcgtccc ggcgtgctgg ccgatctgga
      901 cggcacggtg gacgtggtgg tgtcgaaccc gccgttcgtg cccgagcagg cgcagcttcc
      961 gcccgagttc gcggtgcacc agccgcggat cgccgtcttc gccggcggcg acggcctgga
     1021 ggtgatccgc ggcgtcatcg acaccgcggc ccgactgctg cggcccggcg gccggctcgt
     1081 tctggaacac ggccacctgc acggcgagac gatgccggcg ctgttcgccg cggacgaccg
     1141 cttcacggac atcgcgctgc acaccgacca gttcggctac ccgcactacg tggtcgccac
     1201 ccgcgtctga tcaggacgcg tcgtccatca agcttgcggc cgcactcgag caccaccacc
     1261 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     1321 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     1381 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     1441 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1501 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1561 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1621 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1681 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1741 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     1801 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     1861 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1921 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     1981 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     2041 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     2101 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     2161 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     2221 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     2281 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     2341 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     2401 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     2461 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     2521 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     2581 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     2641 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     2701 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     2761 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     2821 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2881 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2941 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3001 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3061 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3121 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3181 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3241 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3301 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3361 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3421 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3481 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3541 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3601 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3661 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3721 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3781 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3841 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3901 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     3961 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4021 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4081 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4141 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4201 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4261 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4321 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4381 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4441 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4501 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4561 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     4621 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     4681 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     4741 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     4801 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     4861 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     4921 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4981 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     5041 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     5101 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     5161 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     5221 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     5281 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     5341 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     5401 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     5461 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     5521 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     5581 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     5641 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     5701 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     5761 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     5821 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     5881 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     5941 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     6001 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     6061 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     6121 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatg
//
