LOCUS       sequence-389164-        5926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for producing SpyTagged stalled secretion variant
            of intimin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5926)
  AUTHORS   Duodu R, Linke D, Leo JC
  TITLE     Fluorescent Labeling of Outer Membrane Proteins Using the 
            SpyCatcher-SpyTag System.
  JOURNAL   Methods Mol Biol. 2024;2778:53-63. doi: 10.1007/978-1-0716-3734-0_4.
  PUBMED    38478271
REFERENCE   2  (bases 1 to 5926)
  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: "Methods Mol
            Biol."; date: "2024"; volume: "2778:53-63. doi"; pages: " 
            10.1007/978-1-0716-3734-0_4"
COMMENT     Sequence Label: pIBA2-Int-SpyTag
FEATURES             Location/Qualifiers
     source          1..5926
                     /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             2908..2946
                     /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"
     rep_origin      3037..3492
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3124..3143)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3334..3355
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3519..3623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3624..3692,3693..4484)
                     /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(3842..3861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             4491..5117
                     /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(4567..4586)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     rep_origin      5270..5858
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5759..5778
                     /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 caatggtgaa aattatttta aattgggttc ggattcaaaa
      241 ctgttaactc ataatagcta tcagaatcgc cttttttata cgttgaaaac aggtgaaact
      301 gttgccgatc tttctaaatc gcaagatatt aatttatcga cgatttggtc gttgaataag
      361 catttataca gttctgaaag cgaaatgatg aaggccgcgc ctggtcagca gatcattttg
      421 ccactcaaaa aacttccctt tgaatacagt gccttaccac ttttaggttc ggcacctctt
      481 gttgctgcag gtggtgtcgc tggtcataca aataaactga ctaaaatgtc cccggacgtg
      541 accaaaagca acatgaccga tgacaaggca ttaaattatg cggcacaaca ggcggcgagt
      601 ctcggtagcc agcttcagtc gcgatctctg aacggcgatt acgcgaaaga taccgctctt
      661 ggtatcgctg gtaaccaggc ttcgtcacag ttgcaggcct ggttacaaca ttatggaacg
      721 gcagaggtta atctgcagag tggtaataac tttgacggta gttcactgga cttcttatta
      781 ccgttctatg attccgaaaa aatgctggca tttggtcagg tcggagcgcg ttacattgac
      841 tcccgcttta cggcaaattt aggtgcgggt cagcgttttt tccttcctga aaatatgttg
      901 ggctataacg tcttcattga tcaggatttt tctggtgata atacccgttt aggtattggt
      961 ggcgaatact ggcgagacta tttcaaaagt agtgttaacg gctatttccg catgagcggc
     1021 tggcatgagt catacaataa gaaagactat gatgagcgcc cagcaaatgg cttcgatatc
     1081 cgttttaatg gctatctgcc atcatacccg gcattaggtg ccaagctgat gtatgagcag
     1141 tattatggtg ataatgttgc tttgtttaat tctgataagc tgcagtcgaa tcctggtgcg
     1201 gcgaccgttg gtgtaaacta tactccgatt cctctggtga cgatggggat cgattaccgt
     1261 catggtacgg gtaatgaaaa tgatctcctt tactcaatgc agttccgtta tcagtttgat
     1321 aaaccgtggt cccagcaaat tgagccacaa tatgttaacg agttaagaac attatcaggc
     1381 agccgttacg atctggttca gcgtaataac aatattattc tggagtacaa aaagcaggat
     1441 attctttctc tgaatattcc gcatgatatt aatggtactg aacgcagtac gcagaagatt
     1501 caattgatcg ttaagagcaa atacggtctg gatcgtatcg tctgggatga tagtgcatta
     1561 cgtagccagg gcggccagat tcagcatagc ggaagccaaa gcgcacaaga ttaccaggct
     1621 attttgcctg cttatgtgca aggtggtagc aatgtttata aagtgacggc tcgcgcctat
     1681 gaccgtaatg gcaatagctc taacaatgta ctgcttacta ttaccgttct gtcgaatggt
     1741 caggtggtcg accaggttgg ggtaacggac tttacggctg ataagacttc ggctaaagcg
     1801 gatggcaccg aagcaattac ttatactgcg acggtgaaaa agaatggggt agctcaggct
     1861 aatgtccctg tttcatttaa tattgtttca ggaactgcag ttttaagtgc caatagtgcc
     1921 aataccaatg gtagcggtaa ggcgactgta accctgaaat cggataaacc aggccaggtc
     1981 gtcgtgtctg ctaaaacagc agagatgact tcagcgctta atgccaatgc agttatattt
     2041 gttgatcaaa ccaaggccag cattactgag attaaggctg ataaaacaac ggcagtagca
     2101 aatggtcagg atgctattac atacactgtt aaagtgatga agggggataa gcctgtatct
     2161 aatcaggaag tgacctttac gacgacctta ggtaagttaa gtaattccac tgaaaaaacg
     2221 gatacgaatg gctatgccaa agtaacatta acatcgacaa ctccaggaaa atcactcgtt
     2281 agtgcccgtg ttagcgatgt cgccgttgat gtcaaagcac ctgaagttga attttttaca
     2341 acgcttacaa ttgatgacgg taatattgaa attgttggaa ccggagttaa agggaagtta
     2401 cccactgtat ggttgcaata tggtcaagtt aatctgaaag ccagcggagg taacggaaaa
     2461 tatacatggc gctcagcaaa tccagcaatt gcttcggtgg atgcttcttc tggtcaggtc
     2521 accttaaaag agaagggaac tacaactatt tccgttatct caagtgataa tcaaactgca
     2581 acttatacta ttgcaacacc taatagtctg attgttccta atatgagcaa gcgtgtgacc
     2641 tataatgatg ctgtgaatac atgtaagaat tttggaggaa agttgccgtc ttctcagaat
     2701 gaactggaaa atgtctttaa agcatggggg gctgcaaata aatatgaata ttataagtct
     2761 agtcagacta taatttcatg ggtacaacaa acagctcaag atgcgaagag tggtgttgca
     2821 agtacatacg atttagttaa acaaaaccct ctgaataata ttaaggctag tgaatctaat
     2881 gcttatgcca cttgtgtaaa aggtggcgcc catattgtta tggtggatgc atataaaccg
     2941 accaaataat aagcttgacc tgtgaagtga aaaatggcgc acattgtgcg acattttttt
     3001 tgtctgccgt ttaccgctac tgcgtcacgg atctccacgc gccctgtagc ggcgcattaa
     3061 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3121 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3181 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3241 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3301 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3361 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3421 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3481 cgcttacaat ttcaggtggc acttttcggg gaaatgtgct cggaacccct atttgtttat
     3541 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3601 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3661 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3721 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3781 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3841 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3901 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3961 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4021 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4081 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4141 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4201 ctggcgaact acttactcta gcttcccggc aacaattgat agactggatg gaggcggata
     4261 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4321 ctggagccgg tgagcgtggc tctcgcggta tcattgcagc actggggcca gatggtaagc
     4381 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4441 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaggaatta atgatgtctc
     4501 gtttagataa aagtaaagtg attaacagcg cattagagct gcttaatgag gtcggaatcg
     4561 aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct acattgtatt
     4621 ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg ttagataggc
     4681 accatactca cttttgccct ttagaagggg aaagctggca agatttttta cgtaataacg
     4741 ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta catttaggta
     4801 cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt ttatgccaac
     4861 aaggtttttc actagagaat gcattatatg cactcagcgc agtggggcat tttactttag
     4921 gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa acacctacta
     4981 ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac caaggtgcag
     5041 agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa caacttaaat
     5101 gtgaaagtgg gtcttaaaag cagcataacc tttttccgtg atggtaactt cactagttta
     5161 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5221 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5281 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5341 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5401 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5461 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5521 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5581 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5641 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5701 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5761 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5821 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5881 tacggttcct ggccttttgc tggccttttg ctcacatgac ccgaca
//
