LOCUS       sequence-207599-        4234 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For production of SpyCatcher protein fused N-terminally to sfGFP. 
            Includes N-terminal His tag for purification and C-terminal StrepII 
            tag for detection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4234)
  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 4234)
  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: pIBA3-SpyCatcher-sfGFP
FEATURES             Location/Qualifiers
     source          1..4234
                     /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"
     CDS             142..159
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             169..507
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     CDS             514..1224
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             1231..1254
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     rep_origin      1345..1800
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1432..1451)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1642..1663
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1827..1931
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1932..2000,2001..2792)
                     /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(2150..2169)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             2799..3425
                     /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(2875..2894)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     rep_origin      3578..4166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4067..4086
                     /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 gcaccatcac catcaccatg gcgccatggt tgatacctta
      181 tcaggtttat caagtgagca aggtcagtcc ggtgatatga caattgaaga agatagtgct
      241 acccatatta aattctcaaa acgtgatgag gacggcaaag agttagctgg tgcaactatg
      301 gagttgcgtg attcatctgg taaaactatt agtacatgga tttcagatgg acaagtgaaa
      361 gatttctacc tgtatccagg aaaatataca tttgtcgaaa ccgcagcacc agacggttat
      421 gaggtagcaa ctgctattac ctttacagtt aatgagcaag gtcaggttac tgtaaatggc
      481 aaagcaacta aaggtgacgc tcatattggc ggttcaaaag gtgaagaatt atttacaggt
      541 gtagtaccaa ttttagtaga attagatggt gatgtaaatg gtcataaatt ttcagtacgt
      601 ggtgaaggtg aaggtgatgc aacaaatggt aaattaacat taaaattcat ctgtacaaca
      661 ggtaaattac cagtaccatg gccaacatta gtaacaacat taacatatgg tgtacaatgt
      721 ttttcacgtt atccagatca tatgaaacgt catgattttt ttaaatcagc aatgcctgaa
      781 ggttatgtac aagaacgtac aatttcattt aaagatgatg gtacatataa aacacgtgct
      841 gaagtaaaat ttgaaggtga tacattagta aatcgtattg aattaaaagg tattgatttc
      901 aaagaagatg gtaacatttt aggtcataaa ttagaatata acttcaactc acataacgta
      961 tatatcacag cagataaaca aaaaaatggt atcaaagcaa actttaaaat tcgtcataat
     1021 gtagaagatg gttcagtaca attagcagat cattatcaac aaaatacacc aattggtgat
     1081 ggtccagtat tattaccaga taatcattat ttatcaacac aatcagtatt atcaaaagat
     1141 ccaaatgaaa aacgtgatca tatggtatta ttagaatttg taacagcagc aggtattaca
     1201 catggtatgg atgaattata taaaagcgct tggagccacc cgcagttcga aaaataataa
     1261 gcttgacctg tgaagtgaaa aatggcgcac attgtgcgac attttttttg tctgccgttt
     1321 accgctactg cgtcacggat ctccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1381 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1441 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1501 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1561 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1621 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1681 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1741 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1801 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1861 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1921 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1981 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     2041 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2101 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     2161 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2221 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2281 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2341 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2401 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2461 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2521 ttactctagc ttcccggcaa caattgatag actggatgga ggcggataaa gttgcaggac
     2581 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2641 agcgtggctc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2701 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2761 agataggtgc ctcactgatt aagcattggt aggaattaat gatgtctcgt ttagataaaa
     2821 gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa
     2881 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa
     2941 ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact
     3001 tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta
     3061 gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag
     3121 aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac
     3181 tagagaatgc attatatgca ctcagcgcag tggggcattt tactttaggt tgcgtattgg
     3241 aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc
     3301 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct
     3361 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt
     3421 cttaaaagca gcataacctt tttccgtgat ggtaacttca ctagtttaaa aggatctagg
     3481 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3541 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3601 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3661 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3721 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3781 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3841 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3901 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3961 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4021 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4081 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4141 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4201 ccttttgctg gccttttgct cacatgaccc gaca
//
