LOCUS       sequence-324202-        5513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of XMod-Doc mutant XL2V (All LEUs were mutated to VAL in 
            XMod region) with His6x tag and SpyTag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5513)
  AUTHORS   Yang B, Liu H, Liu Z, Doenen R, Nash MA
  TITLE     Influence of Fluorination on Single-Molecule Unfolding and Rupture 
            Pathways of a Mechanostable Protein Adhesion Complex.
  JOURNAL   Nano Lett. 2020 Nov 16. doi: 10.1021/acs.nanolett.0c04178.
  PUBMED    33191756
REFERENCE   2  (bases 1 to 5513)
  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: "Nano Lett. 
            2020 Nov 16. doi: 10.1021/acs.nanolett.0c04178."
COMMENT     Sequence Label: pQE80L-His-SpyTag-XMod(XL2V)-Doc
FEATURES             Location/Qualifiers
     source          1..5513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..15,16..21,22..38,39..45)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    21..37
                     /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)."
     protein_bind    53..69
                     /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             88..99
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             121..138
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             145..183
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     terminator      961..1055
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1099..1758
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1165..1184)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1823..1909
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2002..3084)
                     /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     3046..3065
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3085..3162)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(3246..3268)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3354..3494
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3409..3428
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3509..3526)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3680..4268)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3760..3779)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4439..5230,5231..5299))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5062..5081
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5300..5404)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5472..5490
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcataaaaaa tttatttgct ttgtgagcgg ataacaatta taatagattc aattgtgagc
       61 ggataacaat ttcacacaga attcattaaa gaggagaaat taactatgaa aggcagcagc
      121 catcatcatc atcatcacgt cgacgcccat attgtcatgg ttgatgcata caagccgacg
      181 aagctcgacg gccacaatac cgttaccagc gcagttaaaa cccagtatgt ggaaattgaa
      241 agcgtggatg gcttttattt caacaccgag gataaatttg ataccgcaca gatcaaaaaa
      301 gccgttgttc ataccgtgta taacgaaggt tataccggtg atgatggtgt tgcagttgtt
      361 gttcgtgaat atgaaagcga accggttgat atcacagccg aagttacctt tggtgatgca
      421 acaccggcaa atacctataa agcagtggaa aacaaattcg actatgagat cccggtgtat
      481 tataacaatg ccaccgttaa agatgccgaa ggtaatgatg caaccgtgac cgtttatatt
      541 ggtgttaaag gtgataccga cctgaacaac attgttgatg gtcgtgatgc caccgcaaca
      601 ctgacctatt atgcagcaac cagcaccgat ggtaaagatg cgaccaccgt tgcactgagc
      661 ccgagcacac tggttggtgg taatccggaa agcgtttatg atgatttttc agcatttctg
      721 agcgacgtta aagttgatgc aggtaaagaa ctgacccgct ttgcaaaaaa agcagaacgt
      781 ctgatcgatg gtcgcgacgc aagcagcatt ctgacctttt ataccaaaag cagcgtggat
      841 cagtataaag atatggcagc aaatgaaccg aacaaactgt gggatattgt tacaggtgat
      901 gcggaagaag aataaggatc cggctgctaa caaagcccga agcttaatta gctgagcttg
      961 gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc
     1021 tcggttgccg ccgggcgttt tttattggtg agaatccaag ccagcttggc gagattttca
     1081 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc
     1141 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac
     1201 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     1261 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaatttcgt
     1321 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     1381 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     1441 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     1501 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     1561 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc
     1621 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
     1681 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     1741 tggcagggcg gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggggta
     1801 atgactctct agcttgaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     1861 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccctctag
     1921 attacgtgca gtcgatgata agctgtcaaa catgagaatt gtgcctaatg agtgagctaa
     1981 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     2041 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     2101 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     2161 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     2221 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     2281 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     2341 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     2401 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     2461 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     2521 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     2581 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     2641 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     2701 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     2761 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     2821 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     2881 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     2941 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     3001 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     3061 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     3121 tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg tcggaatttc gggcagcgtt
     3181 gggtcctggc cacgggtgcg catgatctag agctgcctcg cgcgtttcgg tgatgacggt
     3241 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     3301 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     3361 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     3421 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     3481 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3541 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3601 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3661 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3721 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3781 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3841 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3901 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3961 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4021 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4081 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     4141 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4201 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4261 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4321 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4381 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4441 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4501 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4561 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4621 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4681 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4741 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4801 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4861 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4921 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4981 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5041 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5101 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5161 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5221 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5281 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5341 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5401 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     5461 taacctataa aaataggcgt atcacgaggc cctttcgtct tcaccttgag aaa
//
