LOCUS       sequence-198460-        6346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ClfB N2 and N3 domains with C-terminal ybbr tag for covalent 
            immobilization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6346)
  AUTHORS   Milles LF, Schulten K, Gaub HE, Bernardi RC
  TITLE     Molecular mechanism of extreme mechanostability in a pathogen 
            adhesin.
  JOURNAL   Science. 2018 Mar 30;359(6383):1527-1533. doi: 
            10.1126/science.aar2094.
  PUBMED    29599244
REFERENCE   2  (bases 1 to 6346)
  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"; doi: "10.1126/science"; 
            journalName: "Science"; date: "2018-03-30- 30"; volume: "359"; 
            issue: "6383"; pages: "1527-1533"
COMMENT     Sequence Label: pET28a-ClfB_N2N3-HIS-ybbr
FEATURES             Location/Qualifiers
     source          1..6346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(27..45)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      41..88
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      125..580
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(212..231)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     422..443
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(672..1487)
                     /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(672..1487)
                     /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     1395..1414
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1479..1498
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1609..2197
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2098..2117
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2351..2368
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2383..2522
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2448..2467)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2608..2630
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2624..2815)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3624..4706)
                     /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     4668..4687
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4707..4784)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4990..5009
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5093..5112
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5093..5111
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5112..5136
                     /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             5151..5173
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6231..6248
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6255..6278
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
       61 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
      121 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
      181 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
      241 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
      301 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
      361 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
      421 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
      481 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
      541 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
      601 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
      661 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
      721 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
      781 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
      841 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
      901 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
      961 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     1021 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     1081 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     1141 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     1201 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     1261 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     1321 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     1381 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     1441 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     1501 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     1561 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     1621 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     1681 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     1741 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     1801 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     1861 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     1921 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     1981 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     2041 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     2101 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     2161 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     2221 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     2281 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     2341 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     2401 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     2461 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     2521 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     2581 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     2641 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     2701 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     2761 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     2821 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     2881 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     2941 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     3001 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     3061 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     3121 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     3181 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     3241 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     3301 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     3361 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     3421 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     3481 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     3541 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     3601 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3661 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     3721 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     3781 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     3841 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     3901 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     3961 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     4021 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     4081 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     4141 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     4201 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     4261 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     4321 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     4381 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     4441 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     4501 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     4561 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     4621 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     4681 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     4741 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     4801 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     4861 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     4921 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     4981 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     5041 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
     5101 ctcactatag gggaattgtg agcggataac aattcccctg tagaaataat tttgtttaac
     5161 tttaagaagg agatatacat atgggtaccc cggttgttaa tgcagcagat gcaaaaggca
     5221 ccaatgtgaa tgataaagtt accgccagca actttaaact ggaaaaaacc acctttgatc
     5281 cgaatcagag cggtaatacc tttatggcag caaactttac cgttaccgat aaagtgaaaa
     5341 gcggtgatta tttcaccgca aaactgccgg atagcctgac cggtaatggt gatgttgatt
     5401 atagcaatag caataacacc atgccgatcg cagatatcaa aagtaccaac ggtgatgtgg
     5461 ttgcaaaagc cacctatgat attctgacca aaacctatac cttcgtgttc accgattacg
     5521 tgaacaacaa agaaaacatc aatggccagt ttagcctgcc gctgtttacc gatcgtgcaa
     5581 aagcaccgaa aagtggtaca tatgatgcca acattaacat tgccgatgag atgttcaaca
     5641 acaaaatcac ctataactat agcagcccga ttgccggtat tgataaaccg aatggtgcaa
     5701 atattagcag ccagattatt ggtgttgata ccgcaagtgg ccagaatacc tataaacaga
     5761 ccgtttttgt taatccgaaa cagcgtgttc tgggcaatac ctgggtttat atcaaaggct
     5821 atcaggataa aatcgaagag agcagtggta aagttagcgc aaccgatacc aaactgcgta
     5881 tttttgaagt taacgacacc agcaaactga gcgatagcta ttatgcagat ccgaatgata
     5941 gcaacctgaa agaagttacc gaccaattca aaaaccgcat ctattatgaa cacccgaatg
     6001 ttgccagcat caaatttggc gatattacca aaacgtatgt ggttctggtt gaaggccatt
     6061 atgataacac cggcaaaaat ctgaaaacac aggtgattca agaaaacgtt gatccggtta
     6121 ccaaccgtga ttattcaatt tttggctgga acaacgaaaa cgtggttcgt tatggtggtg
     6181 gtagcgcaga tggtgatagt gcagttgagc tcaagcttcc taggtctagg caccaccacc
     6241 accaccacgg atccctggaa gttctgttcc agggtccgga ctctctggaa ttcatcgctt
     6301 ctaaactggc ttaactcgag taagatccgg ctgctaacaa agcccg
//
