LOCUS       sequence-297106-        5670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of E. coli AcpS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5670)
  AUTHORS   Nemec AA, Tomko RJ Jr
  TITLE     A suite of PCR-based peptide tagging plasmids to permit 
            epitope-targeted enzymatic functionalization of yeast proteins.
  JOURNAL   Yeast. 2020 May 13. doi: 10.1002/yea.3471.
  PUBMED    32401365
REFERENCE   2  (bases 1 to 5670)
  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: "Yeast. 2020
            May 13. doi: 10.1002/yea.3471."
COMMENT     Sequence Label: pET28b-6His-AcpS
FEATURES             Location/Qualifiers
     source          1..5670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             535..552
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(605..623)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      619..666
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      703..1158
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(790..809)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1000..1021
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1250..2065)
                     /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(1250..2065)
                     /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     1973..1992
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2057..2076
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2187..2775
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2676..2695
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2929..2946
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2961..3100
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3026..3045)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3186..3208
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3202..3393)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4202..5284)
                     /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     5246..5265
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5285..5362)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5568..5587
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg gcaatattag gtttaggcac ggatattgtg
      181 gagatcgctc gcatcgaagc ggtgatcgcc cgatccggtg atcgcctggc acgccgcgta
      241 ttaagcgata acgaatgggc tatctggaaa acgcaccacc agccggtgcg ttttctggcg
      301 aagcgttttg ctgtgaaaga agccgcagca aaagcgtttg gcaccgggat ccgcaatggt
      361 ctggcgttta atcaatttga agtattcaat gatgagctcg gcaaaccacg gctacggcta
      421 tggggcgagg cattaaaact ggcggaaaag ctgggcgttg caaatatgca tgtaacgctg
      481 gcagatgagc ggcactatgc ttgtgccacg gtaattattg aaagttaact cgagcaccac
      541 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
      601 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
      661 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
      721 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      781 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      841 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      901 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
      961 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     1021 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     1081 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     1141 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1201 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     1261 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     1321 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     1381 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     1441 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     1501 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     1561 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     1621 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     1681 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     1741 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     1801 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     1861 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     1921 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     1981 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     2041 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     2101 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2161 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2221 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2281 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     2341 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2401 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2461 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2521 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2581 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     2641 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     2701 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     2761 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     2821 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     2881 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     2941 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3001 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3061 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3121 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3181 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     3241 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     3301 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     3361 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     3421 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     3481 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     3541 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     3601 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     3661 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     3721 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     3781 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     3841 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     3901 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     3961 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     4021 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     4081 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     4141 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     4201 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4261 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     4321 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     4381 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     4441 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca
     4501 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     4561 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     4621 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     4681 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     4741 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     4801 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     4861 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     4921 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     4981 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     5041 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     5101 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     5161 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     5221 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     5281 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     5341 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     5401 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     5461 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     5521 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     5581 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     5641 tagaggatcg agatctcgat cccgcgaaat
//
