LOCUS       sequence-75396-e        6599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous expression of E. coli CoA biosynthetic enzyme, 
            PPCS_PPCDC - phosphopantothenoylcysteine 
            synthetase_phosphopantothenoylcysteine decarboxylase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6599)
  AUTHORS   Strauss E, Kinsland C, Ge Y, McLafferty FW, Begley TP
  TITLE     Phosphopantothenoylcysteine synthetase from Escherichia coli. 
            Identification and characterization of the last unidentified 
            coenzyme A biosynthetic enzyme in bacteria.
  JOURNAL   J Biol Chem. 2001 Apr 27;276(17):13513-6. Epub 2001 Mar 13.
  PUBMED    11278255
REFERENCE   2  (bases 1 to 6599)
  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"; journalName: "J Biol 
            Chem."; date: "2001-04-27"; volume: "276(17)"; pages: "13513-6. Epub
            2001 Mar 13"
COMMENT     Sequence Label: pET28a-Ec.coaBC (pCLK1210)
FEATURES             Location/Qualifiers
     source          1..6599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..34
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(54..131)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     337..356
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     440..459
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        440..458
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    459..483
                     /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             498..520
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             539..556
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             566..583
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1899..1916
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1969..1987)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1983..2030
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2067..2522
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2154..2173)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2364..2385
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2615..3430)
                     /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(2615..3430)
                     /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     3338..3357
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3422..3441
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3552..4140
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4041..4060
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4294..4311
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4326..4468
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4394..4413)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4554..4576
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4570..4761)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5570..6599,1..53))
                     /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 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
       61 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
      121 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
      181 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
      241 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
      301 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
      361 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
      421 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
      481 tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca
      541 tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatga gcctggccgg
      601 taaaaaaatc gttctcggcg ttagcggcgg tattgctgcc tataaaaccc ctgaactggt
      661 gcgtcgtttg cgcgatcgcg gggccgacgt ccgcgtagcc atgaccgaag cggcaaaagc
      721 ctttatcacc ccacttagct tgcaggcggt ttctggttat cccgtttccg acagtctgct
      781 ggacccggca gccgaagccg ctatgggcca tattgagctg ggtaaatggg ctgatttagt
      841 gattctcgcc cctgccacgg cagatttgat tgcccgtgtt gctgccggaa tggcgaatga
      901 cctggtatcg acgatttgtc tggctacacc tgcgcctgta gccgtgctcc ccgccatgaa
      961 ccagcagatg taccgtgccg ctgccacgca gcataattta gaggtgcttg cttcccgtgg
     1021 tttgctcatc tgggggccag acagtggcag tcaggcttgt ggtgatatcg gtcctgggcg
     1081 aatgctcgat ccgttaacca ttgtggatat ggcggtagcg catttttcgc ccgtcaacga
     1141 cctgaaacat ctgaacatta tgattaccgc cggcccgacg cgtgaaccgc tcgatccggt
     1201 gcgttatatc tctaatcaca gctccggcaa gatgggtttt gctatcgccg ccgccgctgc
     1261 ccgtcgtggc gcgaacgtca cgctggtatc aggtccggtt tcactaccga cgccaccgtt
     1321 tgttaaacgt gttgatgtga tgaccgcgct ggaaatggaa gccgccgtga atgcttctgt
     1381 acagcagcaa aatattttta tcggctgcgc cgccgtggcg gattatcgcg cagctaccgt
     1441 ggccccagag aaaatcaaaa agcaggccac gcagggtgat gaattaacaa taaaaatggt
     1501 taaaaacccc gatatcgtcg caggcgttgc cgcactaaaa gaccatcgac cctacgtcgt
     1561 tggatttgcc gccgaaacaa ataatgtgga agaatacgcc cggcaaaaac gtatccgtaa
     1621 aaaccttgat ctgatctgcg cgaacgatgt ttcccagcca actcaaggat ttaacagcga
     1681 caacaacgca ttacaccttt tctggcagga cggagataaa gtcttaccgc ttgagcgcaa
     1741 agagctcctt ggccaattat tactcgacga gatcgtgacc cgttatgatg aaaaaaatcg
     1801 acgttaagat tctggacccg cgcgttggga aggaatttcc gctcccgact tatgccacct
     1861 ctggctctgc cggacttgac ctgcgtgcct gtctcgagca ccaccaccac caccactgag
     1921 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     1981 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     2041 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2101 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2161 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2221 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2281 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2341 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2401 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2461 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     2521 ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2581 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     2641 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     2701 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     2761 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     2821 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     2881 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     2941 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     3001 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     3061 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     3121 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     3181 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     3241 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     3301 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     3361 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     3421 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     3481 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3541 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3601 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3661 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3721 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3781 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3841 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3901 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3961 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4021 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4081 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4141 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4201 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4261 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4321 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatat
     4381 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     4441 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4501 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4561 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     4621 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4681 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4741 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     4801 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     4861 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4921 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4981 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     5041 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     5101 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     5161 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     5221 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     5281 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     5341 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     5401 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     5461 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     5521 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     5581 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5641 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     5701 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     5761 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     5821 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     5881 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5941 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     6001 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     6061 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     6121 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     6181 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     6241 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     6301 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     6361 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     6421 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     6481 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     6541 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtc
//
