LOCUS       sequence-440343-        6406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E. coli Beta sliding clamp with a His-tag in bacterial 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6406)
  TITLE     Differences between bacteria and eukaryotes in clamp loader 
            mechanism, a conserved process underlying DNA replication
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6406)
  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            "Unpublished"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 Beta sliding clamp (dnaN) in pet28PP
FEATURES             Location/Qualifiers
     source          1..6406
                     /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             3566..3589
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4712..4729
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4782..4800)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4796..4843
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4880..5336
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4967..4986)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5178..5199
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5428..6243)
                     /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(5428..6243)
                     /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     6151..6170
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6235..6254
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(6365..6406,1..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 agggtgattt 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 catcacagca gcggcctgga agttctgttc caggggcccc atatgaaatt
     3601 taccgtagaa cgtgagcatt tattaaaacc gctacaacag gtgagcggtc cgttaggtgg
     3661 tcgtcctacg ctaccgattc tcggtaatct gctgttacag gttgctgacg gtacgttgtc
     3721 gctgaccggt actgatctcg agatggaaat ggtggcacgt gttgcgctgg ttcagccaca
     3781 cgagccagga gcgacgaccg ttccggcgcg caaattcttt gatatctgcc gtggtctgcc
     3841 tgaaggcgcg gaaattgccg tgcagctgga aggtgaacgg atgctggtac gctccgggcg
     3901 tagccgtttt tcgctgtcta ccctgccagc ggcggatttc ccgaacctcg atgactggca
     3961 gagtgaagtc gaatttaccc tgccgcaggc aacgatgaag cgtctgattg aagcgaccca
     4021 gttttctatg gcgcatcagg acgttcgcta ttacttaaat ggtatgctgt ttgaaaccga
     4081 aggtgaagaa ctgcgcaccg tggcaaccga cggccaccgt ctggcggtct gttcaatgcc
     4141 aattggtcaa tctttgccaa gccattcggt gatcgtaccg cgtaaaggcg tgattgaact
     4201 gatgcgtatg ctcgacggcg gcgacaatcc gctgcgcgta cagattggca gcaacaacat
     4261 tcgcgcccac gttggcgact ttatcttcac ctccaaactg gtggatggtc gcttcccgga
     4321 ttatcgccgc gttctgccga agaacccgga caaacatctg gaagctggct gcgatctgct
     4381 caagcaggcg tttgctcgcg cggcgattct ctctaacgag aaattccgcg gcgtacgtct
     4441 ttatgtcagc gaaaaccagc tgaaaatcac cgccaacaac ccggaacagg aagaagcgga
     4501 agagatcctc gacgttacct atagcggtgc ggagatggaa atcggcttca acgtcagtta
     4561 tgtgctggat gttctgaacg cgctgaaatg cgaaaacgtc cgcatgatgc tgaccgattc
     4621 ggtttccagc gtgcagattg aagatgcggc cagccagagc gcggcttatg ttgtcatgcc
     4681 aatgagactg taataagcgg ccgcactcga gcaccaccac caccaccact gagatccggc
     4741 tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     4801 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     4861 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4921 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     4981 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5041 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaaac ttgattaggg
     5101 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5161 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     5221 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     5281 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     5341 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5401 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg
     5461 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     5521 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     5581 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     5641 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     5701 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     5761 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     5821 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     5881 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     5941 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     6001 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     6061 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     6121 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     6181 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
     6241 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
     6301 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6361 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaac
//
