LOCUS       sequence-439388-        6335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli DNA Polymerase III subunit delta' (holb) with a 
            His-tag in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6335)
  TITLE     Differences between bacteria and eukaryotes in clamp loader 
            mechanism, a conserved process underlying DNA replication
  JOURNAL   Unpublished
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: 
            "Unpublished"
COMMENT     Sequence Label: E. coli DNA Polymerase III subunit Delta' (hol...
FEATURES             Location/Qualifiers
     source          1..6335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(798..817)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     958..980
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(974..1165)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1974..3056)
                     /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     3018..3037
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3057..3134)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3340..3359
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3443..3462
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3443..3461
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3462..3486
                     /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             3501..3523
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3542..3559
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3569..3592
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4642..4659
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4712..4730)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4726..4773
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4810..5265
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4897..4916)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5107..5128
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5357..6172)
                     /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(5357..6172)
                     /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     6080..6099
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6164..6183
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(6294..6335,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
      841 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
      901 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
      961 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     1021 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     1081 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     1141 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     1201 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     1261 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     1321 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     1381 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     1441 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     1501 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     1561 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     1621 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     1681 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     1741 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     1801 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     1861 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     1921 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     1981 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2041 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     2101 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     2161 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     2221 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     2281 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     2341 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     2401 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     2461 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     2521 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     2581 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     2641 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     2701 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     2761 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     2821 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     2881 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     2941 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     3001 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     3061 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     3121 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     3181 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     3241 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     3301 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     3361 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     3421 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
     3481 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca tgggcagcag
     3541 ccatcatcat catcatcaca gcagcggcct ggaagttctg ttccaggggc cccatatgag
     3601 atggtatcca tggttacgac ctgatttcga aaaactggta gccagctatc aggccggaag
     3661 aggtcaccat gcgctactca ttcaggcgtt accgggcatg ggcgatgatg ctttaatcta
     3721 cgccctgagc cgttatttac tctgccaaca accgcagggc cacaaaagtt gcggtcactg
     3781 tcgtggatgt cagttgatgc aggctggcac gcatcccgat tactacaccc tggctcccga
     3841 aaaaggaaaa aatacgctgg gcgttgatgc ggtacgtgag gtcaccgaaa agctgaatga
     3901 gcacgcacgc ttaggtggtg cgaaagtcgt ttgggtaacc gatgctgcct tactaaccga
     3961 cgccgcggct aacgcattgc tgaaaacgct tgaagagcca ccagcagaaa cttggttttt
     4021 cctggctacc cgcgagcctg aacgtttact ggcaacatta cgtagtcgtt gtcggttaca
     4081 ttaccttgcg ccgccgccgg aacagtacgc cgtgacctgg ctttcacgcg aagtgacaat
     4141 gtcacaggat gcattacttg ccgcattgcg cttaagcgcc ggttcgcctg gcgcggcact
     4201 ggcgttgttt cagggagata actggcaggc tcgtgaaaca ttgtgtcagg cgttggcata
     4261 tagcgtgcca tcgggcgact ggtattcgct gctagcggcc cttaatcatg aacaagctcc
     4321 ggcgcgttta cactggctgg caacgttgct gatggatgcg ctaaaacgcc atcatggtgc
     4381 tgcgcaggtg accaatgttg atgtgccggg cctggtcgcc gaactggcaa accatctttc
     4441 tccctcgcgc ctgcaggcta tactggggga tgtttgccac attcgtgaac agttaatgtc
     4501 tgttacaggc atcaaccgcg agcttctcat caccgatctt ttactgcgta ttgagcatta
     4561 cctgcaaccg ggcgttgtgc taccggttcc tcatctttaa cgaattcgag ctccgtcgac
     4621 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     4681 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     4741 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     4801 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4861 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4921 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4981 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     5041 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5101 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     5161 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5221 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     5281 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5341 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     5401 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     5461 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     5521 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     5581 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     5641 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     5701 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     5761 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     5821 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     5881 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     5941 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     6001 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     6061 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     6121 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     6181 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     6241 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6301 cctttttttc tgcgcgtaat ctgctgcttg caaac
//
