pKEK-2200 vector (V015602)

LOCUS       62056_13360        5688 bp DNA     circular SYN 13-FEB-2021
DEFINITION  Cloning vector pKEK 2200, complete sequence.
ACCESSION   MT855495
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5688)
  AUTHORS   Mejia-Santana A, Lloyd CJ, Klose KE.
  TITLE     New cloning vectors to facilitate quick allelic exchange in 
            gram-negative bacteria
  JOURNAL   Biotechniques (2021) In press
  PUBMED    33492170
REFERENCE   2  (bases 1 to 5688)
  AUTHORS   Mejia-Santana A.
  TITLE     Direct Submission
  JOURNAL   Submitted (05-AUG-2020) Bology, University of Texas at San Antonio, 
            One UTSA CIrcle, San Antonio, TX 78249, USA
REFERENCE   3  (bases 1 to 5688)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Biotechniques (2021) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (05-AUG-2020) Bology, University of Texas at San Antonio, One UTSA 
            CIrcle, San Antonio, TX 78249, USA"
FEATURES             Location/Qualifiers
     source          1..5688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(4..392)
                     /direction=LEFT
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     CDS             complement(1013..1381)
                     /codon_start=1
                     /label=traJ
                     /note="oriT-recognizing protein"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            complement(1414..1523)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     CDS             complement(2492..3148)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             complement(3249..4667)
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        complement(4668..5113)
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     protein_bind    5237..5258
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5273..5303
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5311..5327
                     /label=lac operator
                     /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)."
     primer_bind     5335..5351
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    5370..5426
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(5436..5452)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"