LOCUS       sequence-75333-e        5957 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Heterologous expression of E. coli CoA biosynthetic enzyme, DPCK 
            (dephospho-coenzyme A kinase). Contain 6xHis-tag for nickel affinity
            purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5957)
  AUTHORS   Strauss E, Begley TP
  TITLE     The antibiotic activity of N-pentylpantothenamide results from its 
            conversion to ethyldethia-coenzyme a, a coenzyme a antimetabolite.
  JOURNAL   J Biol Chem. 2002 Dec 13;277(50):48205-9. Epub 2002 Oct 7.
  PUBMED    12372838
REFERENCE   2  (bases 1 to 5957)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Chem."; date: "2002-12-13"; volume: "277(50)"; pages: "48205-9. Epub
            2002 Oct 7"
COMMENT     Sequence Label: pET28a-Ec.coaE (pESC124)
FEATURES             Location/Qualifiers
     source          1..5957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     465..484
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(504..581)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     787..806
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     890..909
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        890..908
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    909..933
                     /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             948..970
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             989..1006
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1016..1033
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1707..1724
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1777..1795)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1791..1838
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1875..2330
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1962..1981)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2172..2193
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2423..3238)
                     /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(2423..3238)
                     /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     3146..3165
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3230..3249
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3360..3948
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3849..3868
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4102..4119
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4134..4276
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4202..4221)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4362..4384
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4378..4569)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5378..5957,1..503))
                     /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"
ORIGIN
        1 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
       61 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
      121 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
      181 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
      241 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
      301 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
      361 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
      421 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
      481 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
      541 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
      601 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
      661 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
      721 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
      781 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
      841 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
      901 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
      961 aagaaggaga tataccatgg gcagcagcca tcatcatcat catcacagca gcggcctggt
     1021 gccgcgcggc agccatatga ggtatatagt tgccttaacg ggaggcattg gcagtggcaa
     1081 gagtaccgtt gccaatgcgt ttgctgatct cggaattaac gtcattgatg ccgatattat
     1141 tgcgcgtcag gtggttgaac caggtgcacc tgcgctacat gccattgctg atcactttgg
     1201 cgctaacatg attgctgctg atggaacatt gcagcgccgg gccttgcgcg agcggatctt
     1261 cgccaacccg gaagagaaaa actggcttaa cgccctgctg catccgctga ttcagcaaga
     1321 gacgcaacac cagatccagc aagctacttc cccctatgta ctgtgggttg tgccattgct
     1381 ggtagaaaac tcactgtata aaaaagcgaa tcgagtgctt gtggtggatg tcagcccaga
     1441 aacgcaactt aagcgcacca tgcagcgcga tgatgtaact cgcgagcatg tcgaacaaat
     1501 ccttgctgct caggcaacgc gcgaagcccg ccttgccgtg gcagatgacg tcattgataa
     1561 taacggcgca ccggatgcta tcgcatcgga tgttgcccgc ctgcacgcac actatttgca
     1621 gcttgcgtcg cagtttgtct cacaggaaaa accgtaatgc ggatccgaat tcgagctccg
     1681 tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta
     1741 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     1801 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     1861 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1921 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1981 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2041 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2101 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2161 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2221 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2281 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt caggtggcac
     2341 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2401 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     2461 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     2521 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     2581 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     2641 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     2701 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     2761 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     2821 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     2881 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     2941 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3001 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3061 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3121 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3181 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3241 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3301 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3361 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3421 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3481 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3541 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3601 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3661 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3721 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3781 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3841 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3901 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3961 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4021 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4081 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4141 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg tgcactctca
     4201 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4261 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4321 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4381 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     4441 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4501 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     4561 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4621 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4681 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4741 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     4801 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     4861 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     4921 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     4981 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     5041 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     5101 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     5161 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     5221 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     5281 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     5341 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5401 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5461 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     5521 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     5581 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     5641 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     5701 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     5761 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     5821 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     5881 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     5941 gatgctccac gcccagt
//
