LOCUS       Exported                5041 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pKSM713, complete sequence.
ACCESSION   U04895
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5041)
  AUTHORS   Maneewannakul S, Maneewannakul K, Ippen-Ihler K.
  TITLE     The pKSM710 vector cassette provides tightly regulated lac and T7lac
            promoters and strategies for manipulating N-terminal protein 
            sequences
  JOURNAL   Plasmid 31 (3), 300-307 (1994)
  PUBMED    8058823
REFERENCE   2  (bases 1 to 5041)
  AUTHORS   Maneewannakul S.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-JAN-1994) Sumit Maneewannakul, Harvard University, 
            Cellular and Developmental Biology, 16 Divinity Avenue, Cambridge, 
            MA 02138, USA
REFERENCE   3  (bases 1 to 5041)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5041
                     /organism="Cloning vector pKSM713"
                     /lab_host="Escherichia coli"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:31863"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      20..36
                     /regulatory_class="promoter"
                     /note="T7 promoter"
     protein_bind    39..63
                     /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        197..227
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     regulatory      197..226
                     /regulatory_class="promoter"
                     /note="lacUV5 promoter"
     protein_bind    235..251
                     /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             335..607
                     /codon_start=1
                     /gene="lacZM15"
                     /product="beta-galactosidase alpha peptide"
                     /label=lacZM15
                     /protein_id="AAA61622.1"
                     /translation="MGDPKLLLEVPHAISSSPGNSLAVVLQRRDWENPGVTQLNRLAAH
                     PPFASWRNSEEARTDRPSQQLRSLIRLLTKPERKLSWLLPPLSNN"
     gene            335..607
                     /gene="lacZM15"
                     /label=lacZM15
     primer_bind     complement(397..413)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      604..651
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     regulatory      609..650
                     /regulatory_class="terminator"
                     /note="T7 terminator; T-phi"
     terminator      704..790
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     regulatory      707..750
                     /regulatory_class="terminator"
                     /note="rrnBT1 terminator"
     regulatory      881..910
                     /regulatory_class="terminator"
                     /note="rrnBT2 terminator"
     terminator      882..909
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     regulatory      1006..1052
                     /regulatory_class="terminator"
                     /note="bacteriophage fd terminator"
     terminator      1009..1057
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     regulatory      1116..1162
                     /regulatory_class="terminator"
                     /note="bacteriophage fd terminator"
     terminator      1119..1167
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      1295..1750
                     /label=bacteriophage f1 origin
                     /note="bacteriophage f1 origin"
     rep_origin      complement(1903..2497)
                     /direction=LEFT
                     /label=pMB1 origin
                     /note="pMB1 origin"
     rep_origin      complement(1909..2497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     regulatory      complement(2595..2622)
                     /regulatory_class="terminator"
                     /gene="trpA"
     gene            complement(2595..2622)
                     /gene="trpA"
                     /label=trpA
     CDS             complement(2643..3437)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=aminoglycoside phosphotransferase
                     /note="kanamycin resistance gene from Tn5"
                     /protein_id="AAA61623.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     protein_bind    3829..3850
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(3863..4945)
                     /codon_start=1
                     /gene="lacI"
                     /product="Lac repressor"
                     /label=lacI
                     /note="GTG start codon"
                     /protein_id="AAA61624.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            complement(3863..4945)
                     /gene="lacI"
                     /label=lacI
     promoter        complement(4946..5023)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."