LOCUS       sequence-212955-        4354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control construct for SpyCatcher experiments. Encodes MBP with 
            N-terminal SpyTag, including OmpA signal peptide for periplasmic 
            targeting..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4354)
  AUTHORS   Chauhan N, Hatlem D, Orwick-Rydmark M, Schneider K, Floetenmeyer M, 
            van Rossum B, Leo JC, Linke D
  TITLE     Insights into the autotransport process of a trimeric 
            autotransporter, Yersinia Adhesin A (YadA).
  JOURNAL   Mol Microbiol. 2019 Jan 1. doi: 10.1111/mmi.14195.
  PUBMED    30600549
REFERENCE   2  (bases 1 to 4354)
  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: "Mol 
            Microbiol. 2019 Jan 1. doi: 10.1111/mmi.14195."
COMMENT     Sequence Label: pIBA2-SpyTag-MBP
FEATURES             Location/Qualifiers
     source          1..4354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..91
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    42..60
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    72..90
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     sig_peptide     139..201
                     /codon_start=1
                     /product="signal peptide from the E. coli outer membrane 
                     protein OmpA"
                     /label=OmpA signal peptide
                     /translation="MKKTAIAIAVALAGFATVAQA"
     CDS             202..240
                     /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"
     CDS             247..1344
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1201..1210
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1318..1341
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1351..1374
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     rep_origin      1465..1920
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1552..1571)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1762..1783
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1947..2051
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2052..2120,2121..2912)
                     /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     complement(2270..2289)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             2919..3545
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(2995..3014)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     rep_origin      3698..4286
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4187..4206
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccatcgaatg gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt
       61 tattttacca ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct
      121 agataacgag ggcaaaaaat gaaaaagaca gctatcgcga ttgcagtggc actggctggt
      181 ttcgctaccg tagcgcaggc cgcccatatt gttatggtgg atgcatataa accgaccaaa
      241 tctagcaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac
      301 ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag
      361 catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
      421 attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa
      481 atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt
      541 tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac
      601 aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa
      661 ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg
      721 ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt
      781 aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg
      841 attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat
      901 aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc
      961 aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc
     1021 gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag
     1081 ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
     1141 ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc
     1201 gccaccatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct
     1261 ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat
     1321 gaagccctga aagacgcgca gactagcgct tggagccacc cgcagttcga aaaataataa
     1381 gcttgacctg tgaagtgaaa aatggcgcac attgtgcgac attttttttg tctgccgttt
     1441 accgctactg cgtcacggat ctccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1501 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1561 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1621 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1681 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1741 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1801 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1861 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1921 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1981 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2041 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     2101 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2161 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2221 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2281 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2341 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2401 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2461 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2521 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2581 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2641 ttactctagc ttcccggcaa caattgatag actggatgga ggcggataaa gttgcaggac
     2701 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2761 agcgtggctc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2821 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2881 agataggtgc ctcactgatt aagcattggt aggaattaat gatgtctcgt ttagataaaa
     2941 gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa
     3001 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa
     3061 ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact
     3121 tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta
     3181 gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag
     3241 aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac
     3301 tagagaatgc attatatgca ctcagcgcag tggggcattt tactttaggt tgcgtattgg
     3361 aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc
     3421 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct
     3481 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt
     3541 cttaaaagca gcataacctt tttccgtgat ggtaacttca ctagtttaaa aggatctagg
     3601 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3661 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3721 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3781 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3841 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3901 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3961 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4021 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4081 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4141 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4201 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4261 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4321 ccttttgctg gccttttgct cacatgaccc gaca
//
