LOCUS       sequence-306454-        5962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression (amber suppression) of Rc. XDocB 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 (WT, S199AzF)-HIS
FEATURES             Location/Qualifiers
     source          1..5962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     935..954
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(974..1051)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1257..1276
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1360..1379
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1360..1378
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1379..1403
                     /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             1418..1440
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2177..2194
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2256..2274)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2270..2317
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2354..2809
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2441..2460)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2651..2672
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2901..3716)
                     /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(2901..3716)
                     /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     3624..3643
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3708..3727
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3838..4426
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4327..4346
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4580..4597
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4612..4751
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4677..4696)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4837..4859
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4853..5044)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5853..5962,1..973))
                     /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 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
       61 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
      121 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
      181 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
      241 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
      301 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
      361 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
      421 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
      481 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
      541 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
      601 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
      661 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
      721 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
      781 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
      841 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
      901 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
      961 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     1021 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     1081 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     1141 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     1201 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     1261 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     1321 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     1381 aattgtgagc ggataacaat tcccctgtag aaataatttt gtttaacttt aagaaggaga
     1441 tatacatatg ggtaccgact ctctggaatt catcgcttct aaactggctg gctctagcgg
     1501 ttccgaaaca actaccgaga cttctgtgtc tcaatctttc tccttcacag agcccactcg
     1561 cgtatgctac tggtcccatg acaaccgtcc tttcgacctg gagggcttga aagtcattct
     1621 tcaggagacg atctttatca tccacgagga cggcacagtc gtcgaagcag aagagcaacc
     1681 cgctgccgta gacatcacag agcagtgtat gactatcttc tgtaacgata agcaagtggc
     1741 gacgccagaa gatacctacg aaacgcagat ctataagtac atgctggagt ttgcatatga
     1801 aggcaacccg gacgttattg tggggactca tcaagtcttg attggtgtga aaggagatac
     1861 aaatctgagc ggcgacgtaa cagttgcaga tgctgttgag atcctgatgt accgtgctga
     1921 catgatggct aatcccaaca acccaacata cgtatttaac gaagaccccg atctgcataa
     1981 attagggtat tttttgggta atgtagattg tatcaaagag ggtgacgatt taaacgttgc
     2041 cgacgccgtc aatatcttaa tgtttcgtgc ggaggtctta gccaacggct aggcaacgtg
     2101 ggagcagatt gttggctatg accttattga cccaagcgac gagagtacct ttcgtactat
     2161 tgagaagctg ggctctcatc atcatcacca tcactaactc gagtaagatc cggctgctaa
     2221 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     2281 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     2341 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2401 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2461 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2521 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2581 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     2641 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     2701 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     2761 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     2821 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2881 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     2941 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     3001 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     3061 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     3121 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     3181 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     3241 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     3301 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     3361 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     3421 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     3481 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     3541 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     3601 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     3661 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     3721 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
     3781 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3841 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3901 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3961 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     4021 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4081 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4141 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4201 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4261 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4321 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4381 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4441 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4501 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4561 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4621 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     4681 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     4741 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4801 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4861 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     4921 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     4981 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     5041 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     5101 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     5161 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     5221 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     5281 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     5341 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     5401 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     5461 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     5521 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     5581 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     5641 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     5701 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     5761 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     5821 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5881 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5941 ggcgccaggg tggtttttct tt
//
