LOCUS       sequence-306484-        5962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression (amber suppression) of Rc. XDocB binding mode B 
            knock-out mutant with serine at position 199 replaced with amber 
            codon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5962)
  AUTHORS   Liu Z, Liu H, Vera AM, Bernardi RC, Tinnefeld P, Nash MA
  TITLE     High force catch bond mechanism of bacterial adhesion in the human 
            gut.
  JOURNAL   Nat Commun. 2020 Aug 28;11(1):4321. doi: 10.1038/s41467-020-18063-x.
  PUBMED    32859904
REFERENCE   2  (bases 1 to 5962)
  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: "Nat 
            Commun."; date: "2020-08-28"; volume: "11(1):4321. doi"; pages: " 
            10.1038/s41467-020-18063-x"
COMMENT     Sequence Label: pET28a-ybbr-XMod-Doc (BMB-KO, S199AzF)-HIS
FEATURES             Location/Qualifiers
     source          1..5962
                     /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             818..835
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(897..915)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      911..958
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      995..1450
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1082..1101)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1292..1313
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1542..2357)
                     /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(1542..2357)
                     /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     2265..2284
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2349..2368
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2479..3067
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2968..2987
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3221..3238
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3253..3392
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3318..3337)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3478..3500
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3494..3685)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4494..5576)
                     /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     5538..5557
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5577..5654)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5860..5879
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggtaccgac tctctggaat tcatcgcttc
      121 taaactggct ggctctagcg gttccgaaac aactaccgag acttctgtgt ctcaatcttt
      181 ctccttcaca gagcccactc gcgtatgcta ctggtcccat gacaaccgtc ctttcgacct
      241 ggagggcttg aaagtcattc ttcaggagac gatctttatc atccacgagg acggcacagt
      301 cgtcgaagca gaagagcaac ccgctgccgt agacatcaca gagcagtgta tgactatctt
      361 ctgtaacgat aagcaagtgg cgacgccaga agatacctac gaaacgcaga tctataagta
      421 catgctggag tttgcatatg aaggcaaccc ggacgttatt gtggggactc atcaagtctt
      481 gattggtgtg aaaggagata caaatctgag cggcgacgta acagttgcag atgctgttga
      541 gatcctgatg tacgctgctg acatggaggc taatcccaac aacccaacat acgtatttaa
      601 cgaagacccc gatctgcata aattagggta ttttttgggt aatgtagatt gtatcaaaga
      661 gggtgacgat ttaaacgttg ccgacgccgt caatatctta atgtttcgtg cggaggtctt
      721 agccaacggc taggcaacgt gggagcagat tgttggctat gaccttattg acccaagcga
      781 cgagagtacc tttcgtacta ttgagaagct gggctctcat catcatcacc atcactaact
      841 cgagtaagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
      901 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
      961 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1021 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1081 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1141 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1201 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1261 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1321 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1381 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1441 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1501 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     1561 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1621 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1681 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     1741 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     1801 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     1861 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     1921 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     1981 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2041 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2101 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2161 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2221 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2281 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2341 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2401 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2461 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2521 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2581 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     2641 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2701 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2761 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2821 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2881 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2941 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3001 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3061 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3121 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3181 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3241 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3301 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3361 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3421 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3481 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3541 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     3601 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     3661 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     3721 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     3781 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     3841 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     3901 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     3961 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4021 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4081 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4141 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4201 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4261 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4321 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4381 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4441 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4501 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4561 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     4621 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     4681 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     4741 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     4801 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     4861 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     4921 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     4981 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5041 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5101 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5161 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5221 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5281 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5341 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5401 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5461 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5521 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     5581 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     5641 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     5701 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     5761 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     5821 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     5881 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     5941 cgagatctcg atcccgcgaa at
//
