LOCUS       Exported                7202 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pKOD_mazF, complete sequence.
ACCESSION   JX644442
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7202)
  AUTHORS   Al-Hinai MA, Fast AG, Papoutsakis ET.
  TITLE     Novel system for efficient isolation of clostridium double-crossover
            allelic exchange mutants enabling markerless chromosomal gene 
            deletions and DNA integration
  JOURNAL   Appl. Environ. Microbiol. 78 (22), 8112-8121 (2012)
  PUBMED    22983967
REFERENCE   2  (bases 1 to 7202)
  AUTHORS   Al-Hinai MA, Papoutsakis ET, Fast AG.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-SEP-2012) Biological Sciences, University of Delaware,
            15 Innovation Way, Newark, DE 19711, USA
REFERENCE   3  (bases 1 to 7202)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7202
                     /organism="Cloning vector pKOD_mazF"
                     /mol_type="other DNA"
                     /db_xref="taxon:1235839"
     CDS             37..696
                     /codon_start=1
                     /gene="catp"
                     /product="chloramphenicol and thiamphenicol resistance 
                     protein"
                     /label=catp
                     /note="CatP"
                     /protein_id="AFS68384.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     gene            37..696
                     /gene="catp"
                     /label=catp
     CDS             1038..1343
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             1083..1343
                     /codon_start=1
                     /gene="ccdb"
                     /product="Ccdb"
                     /label=ccdb
                     /protein_id="AFS68385.1"
                     /translation="MFVDVQSDIIDTPGRRMVIPLASARLLSDKVSRELYPVVHIGDES
                     WRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     gene            1083..1343
                     /gene="ccdb"
                     /label=ccdb
     protein_bind    complement(1384..1508)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1533..1973)
                     /codon_start=1
                     /gene="repL"
                     /product="gram positive replication protein"
                     /label=repL
                     /note="RepL"
                     /protein_id="AFS68389.1"
                     /translation="MKERYGTVYKGSQRLIDEESGEVIEVDKLYRKQTSGNFVKAYIVQ
                     LISMLDMIGGKKLKIVNYILDNVHLSNNTMIATTREIAKATGTSLQTVITTLKILEEGN
                     IIKRKTGVLMLNPELLMRGDDQKQKYLLLEFGNFEQEANEID"
     gene            complement(1533..1973)
                     /gene="repL"
                     /label=repL
     CDS             complement(2584..3423)
                     /codon_start=1
                     /gene="bgaR"
                     /product="beta-galactosidase regulator"
                     /label=bgaR
                     /note="BgaR"
                     /protein_id="AFS68386.1"
                     /translation="MQILWKKYVKENFEMNVDECGIEQGIPGLGYNYEVLKNAVIHYVT
                     KGYGTFKFNGKVYNLKQGDIFILLKGMQVEYVASIDDPWEYYWIGFSGSNANEYLNRTS
                     ITNSCVANCEENSKIPQIILNMCEISKTYNPSRSDDILLLKELYSLLYALIEEFPKPFE
                     YKDKELHTYIQDALNFINSNYMHSITVQEIADYVNLSRSYLYKMFIKNLGISPQRYLIN
                     LRMYKATLLLKSTKLPIGEVASSVGYSDSLLFSKTFSKHFSMSPLNYRNNQVNKPSI"
     gene            complement(2584..3423)
                     /gene="bgaR"
                     /label=bgaR
     protein_bind    3854..3870
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     CDS             3889..4224
                     /codon_start=1
                     /gene="mazf"
                     /product="MazF toxin"
                     /label=mazf
                     /note="codon-optimized"
                     /protein_id="AFS68387.1"
                     /translation="MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGM
                     CLCVPCTTQSKGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKA
                     KINVLIG"
     gene            3889..4224
                     /gene="mazf"
                     /label=mazf
     CDS             complement(4361..5095)
                     /codon_start=1
                     /gene="MLS"
                     /product="erythromycin resistance protein"
                     /label=MLS
                     /protein_id="AFS68388.1"
                     /translation="MNEKNIKHSQNFITSKHNIDKIMTNIRLNEHDNIFEIGSGKGHFT
                     LELVQRCNFVTAIEIDHKLCKTTENKLVDHDNFQVLNKDILQFKFPKNQSYKIFGNIPY
                     NISTDIIRKIVFDSIADEIYLIVEYGFAKRLLNTKRSLALFLMAEVDISILSMVPREYF
                     HPKPKVNSSLIRLNRKKSRISHKDKQKYNYFVMKWVNKEYKKIFTKNQFNNSLKHAGID
                     DLNNISFEQFLSLFNSYKLFNK"
     gene            complement(4361..5095)
                     /gene="MLS"
                     /label=MLS
     primer_bind     complement(5627..5643)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5651..5667
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     promoter        complement(5675..5705)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5720..5741
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(6029..6617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    7006..7130
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        7155..7185
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"