LOCUS       Exported                4598 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pST98-AS, complete sequence.
ACCESSION   AF170483
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4598)
  AUTHORS   Posfai G, Kolisnychenko V, Bereczki Z, Blattner FR.
  TITLE     Markerless gene replacement in Escherichia coli stimulated by a 
            double-strand break in the chromosome
  JOURNAL   Nucleic Acids Res. 27 (22), 4409-4415 (1999)
  PUBMED    10536150
REFERENCE   2  (bases 1 to 4598)
  AUTHORS   Posfai G, Kolisnychenko V, Bereczki Z, Blattner FR.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-JUL-1999) Institute of Biochemistry, Biol. Res. Center
            Hung. Acad.Sci., Temesvari krt.,62, Szeged H-6726, Hungary
REFERENCE   3  (bases 1 to 4598)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4598
                     /organism="Cloning vector pST98-AS"
                     /lab_host="Escherichia coli"
                     /mol_type="genomic DNA"
                     /label=suicide plasmid for gene replacement
                     /note="suicide plasmid for gene replacement"
                     /db_xref="taxon:102132"
     CDS             complement(71..694)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor"
                     /label=tetR
                     /note="derived from transposon Tn10"
                     /protein_id="AAD51643.1"
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     gene            complement(71..694)
                     /gene="tetR"
                     /label=tetR
     promoter        713..768
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     regulatory      714..719
                     /regulatory_class="minus_35_signal"
                     /note="tetA promoter from transposon Tn10"
     protein_bind    719..737
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     misc_binding    729..737
                     /label=TetR binding site
                     /bound_moiety="TetR"
                     /note="site A"
     regulatory      738..744
                     /regulatory_class="minus_10_signal"
                     /note="tetA promoter from transposon Tn10"
     protein_bind    749..767
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_binding    759..767
                     /label=TetR binding site
                     /bound_moiety="TetR"
                     /note="site B"
     CDS             776..1477
                     /codon_start=1
                     /gene="I-SceI"
                     /product="I-SceI meganuclease"
                     /label=I-SceI
                     /protein_id="AAD51644.1"
                     /translation="MHQKNQVMNLGPNSKLLKEYKSQLIELNIEQFEAGIGLILGDAYI
                     RSRDEGKTYCMQFEWKNKAYMDHVCLLYDQWVLSPPHKKQRVNHLGNLVITWGAQTFKH
                     QAFNKLANLFIVNNKKTIPNNLVENYLTPMSLAYWFMDDGGKWDYNKNSTNKSIVLNTQ
                     SFTFEEVEYLVKGLRNKFQLNCYVKINKNKPIIYIDSMSYLIFYNLIKPYLIPQMMYKL
                     PNTISSETFLK"
     gene            776..1477
                     /gene="I-SceI"
                     /label=I-SceI
     promoter        1525..1543
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1542
                     /label=T7 RNA polymerase transcription initiation site
                     /note="T7 RNA polymerase transcription initiation site"
     misc_feature    1551..1635
                     /label=multiple cloning site
                     /note="multiple cloning site"
     misc_feature    1588
                     /label=I-SceI cleavage site
                     /note="I-SceI cleavage site"
     CDS             complement(1764..2714)
                     /codon_start=1
                     /gene="rep ts"
                     /product="Rep ts replication protein"
                     /label=rep ts
                     /note="derived from pHS1"
                     /protein_id="AAD51645.1"
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     gene            complement(1764..2714)
                     /gene="rep ts"
                     /label=rep ts
     rep_origin      2715..3094
                     /label=derived from pHS1
                     /note="derived from pHS1"
     rep_origin      2762..2984
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(3606..4466)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=beta-lactamase
                     /protein_id="AAD51646.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4467..4571)
                     /gene="bla"
                     /label=AmpR promoter