LOCUS       sequence-306456-        5962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression (amber suppression) of Rc. XDocB binding mode A 
            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 (BMA-KO, S199AzF)-HIS
FEATURES             Location/Qualifiers
     source          1..5962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     950..969
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(989..1066)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1272..1291
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1375..1394
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1375..1393
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1394..1418
                     /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             1433..1455
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2192..2209
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2271..2289)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2285..2332
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2369..2824
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2456..2475)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2666..2687
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2916..3731)
                     /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(2916..3731)
                     /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     3639..3658
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3723..3742
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3853..4441
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4342..4361
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4595..4612
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4627..4766
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4692..4711)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4852..4874
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4868..5059)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5868..5962,1..988))
                     /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"
ORIGIN
        1 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
       61 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
      121 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
      181 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
      241 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
      301 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
      361 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
      421 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
      481 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
      541 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
      601 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
      661 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
      721 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
      781 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
      841 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
      901 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
      961 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     1021 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     1081 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     1141 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     1201 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     1261 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     1321 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     1381 gactcactat aggggaattg tgagcggata acaattcccc tgtagaaata attttgttta
     1441 actttaagaa ggagatatac atatgggtac cgactctctg gaattcatcg cttctaaact
     1501 ggctggctct agcggttccg aaacaactac cgagacttct gtgtctcaat ctttctcctt
     1561 cacagagccc actcgcgtat gctactggtc ccatgacaac cgtcctttcg acctggaggg
     1621 cttgaaagtc attcttcagg agacgatctt tatcatccac gaggacggca cagtcgtcga
     1681 agcagaagag caacccgctg ccgtagacat cacagagcag tgtatgacta tcttctgtaa
     1741 cgataagcaa gtggcgacgc cagaagatac ctacgaaacg cagatctata agtacatgct
     1801 ggagtttgca tatgaaggca acccggacgt tattgtgggg actcatcaag tcttgattgg
     1861 tgtgaaagga gatacaaatc tgagcggcga cgtaacagtt gcagatgctg ttgagatcct
     1921 gatgtaccgt gctgacatga tggctaatcc caacaaccca acatacgtat ttaacgaaga
     1981 ccccgatctg cataaattag ggtatttttt gggtaatgta gattgtatca aagagggtga
     2041 cgatttaaac gttgccgacg ccgtcaatat cttaatgttt gctgcggagg tcgaagccaa
     2101 cggctaggca acgtgggagc agattgttgg ctatgacctt attgacccaa gcgacgagag
     2161 tacctttcgt actattgaga agctgggctc tcatcatcat caccatcact aactcgagta
     2221 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     2281 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     2341 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     2401 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2461 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2521 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2581 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2641 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2701 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2761 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     2821 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2881 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     2941 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     3001 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     3061 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     3121 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     3181 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     3241 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     3301 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     3361 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     3421 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     3481 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     3541 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     3601 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     3661 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     3721 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3781 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3841 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3901 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3961 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     4021 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4081 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4141 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4201 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4261 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4321 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4381 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4441 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4501 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4561 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4621 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     4681 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     4741 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     4801 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4861 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     4921 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     4981 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     5041 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     5101 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     5161 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     5221 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     5281 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     5341 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     5401 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     5461 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     5521 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg
     5581 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc
     5641 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg
     5701 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca
     5761 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag
     5821 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
     5881 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5941 gcggtttgcg tattgggcgc ca
//
