LOCUS       sequence-75331-e        6335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous expression of E. coli CoA biosynthetic enzyme, PanK 
            (pantothenate kinase), induced with IPTG. Contain 6xHis-tag for 
            nickel affinity purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6335)
  AUTHORS   Brand LA, Strauss E
  TITLE     Characterization of a new pantothenate kinase isoform from 
            Helicobacter pylori.
  JOURNAL   J Biol Chem. 2005 May 27;280(21):20185-8. Epub 2005 Mar 28.
  PUBMED    15795230
REFERENCE   2  (bases 1 to 6335)
  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: "2005-05-27"; volume: "280(21)"; pages: "20185-8. Epub
            2005 Mar 28"
COMMENT     Sequence Label: pET28a-Ec.coaA
FEATURES             Location/Qualifiers
     source          1..6335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     119..138
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(158..235)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     441..460
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     544..563
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        544..562
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    563..587
                     /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             602..624
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             643..660
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             670..687
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1739..1756
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1809..1827)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1823..1870
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1907..2362
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1994..2013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2204..2225
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2455..3270)
                     /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(2455..3270)
                     /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     3178..3197
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3262..3281
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3392..3980
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3881..3900
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4134..4151
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4166..4308
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4234..4253)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4394..4416
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4410..4601)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5410..6335,1..157))
                     /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 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
       61 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
      121 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
      181 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
      241 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
      301 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
      361 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
      421 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
      481 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
      541 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
      601 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac
      661 agcagcggcc tggtgccgcg cggcagccat atgagtataa aagagcaaac gttaatgacg
      721 ccttacctac agtttgaccg caaccagtgg gcagctctgc gtgattccgt acctatgacg
      781 ttatcggaag atgagatcgc ccgtctcaaa ggtattaatg aagatctctc gttagaagaa
      841 gttgccgaga tctatttacc tttgtcacgt ttgctgaact tctatataag ctcgaatctg
      901 cgccgtcagg cagttctgga acagtttctt ggtactaacg ggcaacgcat tccttacatt
      961 atcagtattg ctggcagtgt cgcggtgggg aaaagtacaa ccgcccgtgt attgcaggcg
     1021 ctattaagcc gttggccgga acatcgtcgt gttgaactga tcactacaga tggcttcctt
     1081 caccctaatc aggttctgaa agaacgtggt ctgatgaaga agaaaggctt cccggaatcg
     1141 tatgatatgc atcgcctggt gaagtttgtt tccgatctca aatccggcgt gccaaacgtt
     1201 acagcccctg tttactcgca tcttatttat gatgtgatcc cggatggaga taaaacggtt
     1261 gttcagcctg atattttaat tcttgaaggg ttaaatgtct tacagagcgg gatggattat
     1321 ccacacgatc cacatcatgt atttgtttct gattttgtcg atttttcgat atatgttgat
     1381 gcaccggaag acttacttca gacgtggtat atcaaccgtt ttctgaaatt ccgcgaaggg
     1441 gcttttaccg acccggattc ctattttcat aactacgcga aattaactaa agaagaagcg
     1501 attaagactg ccatgacatt gtggaaagag atcaactggc tgaacttaaa gcaaaatatt
     1561 ctacctactc gtgagcgcgc cagtttaatc ctgacgaaaa gtgctaatca tgcggtagaa
     1621 gaggtcagac tacgcaaata atttgcaggg gagcgaatac tcccctttct tatttttctg
     1681 cactacgcag ggatatttca ccgcccatcc agggttttat tattccatcc tgctcgagca
     1741 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     1801 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1861 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     1921 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1981 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2041 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2101 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     2161 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2221 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     2281 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     2341 aaaatattaa cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct
     2401 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     2461 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     2521 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     2581 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     2641 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     2701 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     2761 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     2821 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     2881 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     2941 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     3001 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     3061 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     3121 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     3181 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     3241 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     3301 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3361 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3421 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3481 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3541 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3601 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3661 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3721 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3781 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3841 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3901 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3961 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4021 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4081 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4141 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4201 gtatttcaca ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4261 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4321 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4381 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4441 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     4501 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4561 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4621 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4681 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4741 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4801 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4861 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4921 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4981 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     5041 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     5101 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     5161 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     5221 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     5281 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     5341 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     5401 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5461 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     5521 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5581 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     5641 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     5701 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     5761 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     5821 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     5881 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     5941 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     6001 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     6061 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     6121 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     6181 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     6241 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     6301 aatcagcaac gactgtttgc ccgccagttg ttgtg
//
