LOCUS       Exported                4133 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Phagemid cloning vector pKSS, complete sequence.
ACCESSION   U01668
VERSION     .
KEYWORDS    direct-selection cloning vehicle; positive-selection; pheS.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4133)
  AUTHORS   Selzer G, Som T, Itoh T, Tomizawa J.
  TITLE     The origin of replication of plasmid p15A and comparative studies on
            the nucleotide sequences around the origin of related plasmids
  JOURNAL   Cell 32 (1), 119-129 (1983)
  PUBMED    6186390
REFERENCE   2  (bases 1 to 4133)
  AUTHORS   Fayat G, Mayaux JF, Sacerdot C, Fromant M, Springer M, 
            Grunberg-Manago M, Blanquet S.
  TITLE     Escherichia coli phenylalanyl-tRNA synthetase operon region. 
            Evidence for an attenuation mechanism. Identification of the gene 
            for the ribosomal protein L20
  JOURNAL   J. Mol. Biol. 171 (3), 239-261 (1983)
  PUBMED    6317865
REFERENCE   3  (bases 1 to 4133)
  AUTHORS   Yanisch-Perron C, Vieira J, Messing J.
  TITLE     Improved M13 phage cloning vectors and host strains: nucleotide 
            sequences of the M13mp18 and pUC19 vectors
  JOURNAL   Gene 33 (1), 103-119 (1985)
  PUBMED    2985470
REFERENCE   4  (bases 1 to 4133)
  AUTHORS   Balbas P, Soberon X, Merino E, Zurita M, Lomeli H, Valle F, Flores 
            N, Bolivar F.
  TITLE     Plasmid vector pBR322 and its special-purpose derivatives--a review
  JOURNAL   Gene 50 (1-3), 3-40 (1986)
  PUBMED    3034735
REFERENCE   5  (bases 1 to 4133)
  AUTHORS   Short JM, Fernandez JM, Sorge JA, Huse WD.
  TITLE     Lambda ZAP: a bacteriophage lambda expression vector with in vivo 
            excision properties
  JOURNAL   Nucleic Acids Res. 16 (15), 7583-7600 (1988)
  PUBMED    2970625
REFERENCE   6  (bases 1 to 4133)
  AUTHORS   Kast P, Hennecke H.
  TITLE     Amino acid substrate specificity of Escherichia coli 
            phenylalanyl-tRNA synthetase altered by distinct mutations
  JOURNAL   J. Mol. Biol. 222 (1), 99-124 (1991)
  PUBMED    1942071
REFERENCE   7  (bases 1 to 4133)
  AUTHORS   Alting-Mees MA, Sorge JA, Short JM.
  TITLE     pBluescriptII: multifunctional cloning and mapping vectors
  JOURNAL   Meth. Enzymol. 216, 483-495 (1992)
  PUBMED    1362236
REFERENCE   8  (bases 1 to 4133)
  AUTHORS   Lin-Chao S, Chen WT, Wong TT.
  TITLE     High copy number of the pUC plasmid results from a 
            Rom/Rop-suppressible point mutation in RNA II
  JOURNAL   Mol. Microbiol. 6 (22), 3385-3393 (1992)
  PUBMED    1283002
REFERENCE   9  (bases 1 to 4133)
  AUTHORS   Kast P.
  TITLE     pKSS--a second-generation general purpose cloning vector for 
            efficient positive selection of recombinant clones
  JOURNAL   Gene 138 (1-2), 109-114 (1994)
  PUBMED    8125286
REFERENCE   10  (bases 1 to 4133)
  AUTHORS   Kast P.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-SEP-1993) Dept. of Chemistry and Molecular Biology, 
            The Scripps Research Institute, 10550 North Torrey Pines Road, La 
            Jolla, CA 92037, USA
REFERENCE   11  (bases 1 to 4133)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     On Oct 6, 1993 this sequence version replaced gi:403935.
FEATURES             Location/Qualifiers
     source          1..4133
                     /organism="unidentified cloning vector"
                     /lab_host="Escherichia coli"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:45196"
     misc_feature    join(1..55,1240..4133)
                     /label=derived from GenBank accession X52331
                     /note="derived from GenBank accession X52331"
     misc_feature    1..61
                     /note="multiple cloning site I: 
                     KpnI-EcoO109I-ApaI-AvaI-XhoI-SalI-AccI-HincII-ClaI-H 
                     indIII-EcoRV-EcoRI-AvaI-SmaI"
                     /citation="[9]"
     primer_bind     18..34
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             146..1129
                     /codon_start=1
                     /gene="pheS"
                     /product="phenylalanyl-tRNA synthetase alpha subunit 
                     (Gly294 variant)"
                     /function="confers p-Cl-phenylalanine sensitivity"
                     /EC_number="6.1.1.20"
                     /label=pheS
                     /citation="[2]"
                     /citation="[6]"
                     /protein_id="AAB61946.1"
                     /translation="MSHLAELVASAKAAISQASDVAALDNVRVEYLGKKGHLTLQMTTL
                     RELPPEERPAAGAVINEAKEQVQQALNARKAELESAALNARLAAETIDVSLPGRRIENG
                     GLHPVTRTIDRIESFFGELGFTVATGPEIEDDYHNFDALNIPGHHPARADHDTFWFDTT
                     RLLRTQTSGVQIRTMKAQQPPIRIIAPGRVYRNDYDQTHTPMFHQMEGLIVDTNISFTN
                     LKGTLHDFLRNFFEEDLQIRFRPSYFPFTEPSAEVDVMGKNGKWLEVLGCGMVHPNVLR
                     NVGIDPEVYSGFGFGMGMERLTMLRYGVTDLRSFFENDLRFLKQFK"
     gene            146..1129
                     /gene="pheS"
                     /label=pheS
                     /note="Gly294 mutant"
     old_sequence    365..366
                     /gene="pheS"
                     /replace="gc"
                     /citation="[2]"
                     /citation="[6]"
     variation       1026
                     /gene="pheS (wild type)"
                     /replace="c"
                     /note="the exchange of codon GCC (Ala294, wild type) with 
                     GGC (Gly294, mutant pheS, as present on pKSS) yielded the 
                     phenotypic change from p-Cl-phenylalanine resistance to 
                     sensitivity"
                     /citation="[6]"
     gene            1026
                     /gene="pheS (wild type)"
                     /label=pheS (wild type)
     misc_feature    1243..1284
                     /note="multiple cloning site II: 
                     BamHI-SpeI-XbaI-EagI-NotI-DsaI-SacII(KspI)-SacI(SstI)"
                     /citation="[9]"
     primer_bind     complement(1244..1260)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      complement(1292..1311)
                     /regulatory_class="promoter"
                     /standard_name="T7 promoter"
                     /citation="[5]"
                     /citation="[7]"
                     /citation="[9]"
     promoter        complement(1293..1311)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1314..1474
                     /codon_start=1
                     /gene="lacZ"
                     /product="beta-galactosidase fragment"
                     /EC_number="3.2.1.23"
                     /label=lacZ
                     /note="N-truncated alpha-fragment of beta-galactosidase; 
                     blue/white screening with the lacZ system is irrelevant in 
                     pKSS"
                     /citation="[5]"
                     /citation="[7]"
                     /citation="[9]"
                     /protein_id="AAB61947.1"
                     /translation="IHWPSFYNVVTGKTLALPNLIALQHIPLSPAGVIAKRPAPIALPN
                     SCAA*MAN"
     gene            1314..1474
                     /gene="lacZ"
                     /label=lacZ
     primer_bind     complement(1318..1334)
                     /note="annealing site for universal M13(-20) sequencing 
                     primer"
                     /citation="[5]"
                     /citation="[9]"
     rep_origin      1476..1931
                     /standard_name="f1 phage origin region for single strand 
                     DNA replication"
                     /note="nicking site of gene II protein is between pos. 1768
                     and 1769"
                     /citation="[5]"
                     /citation="[7]"
     old_sequence    1922
                     /replace="t"
                     /citation="[5]"
     promoter        1957..2061
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2062..2922
                     /codon_start=1
                     /gene="bla"
                     /product="pre-beta-lactamase"
                     /function="provides resistance to ampicillin"
                     /EC_number="3.5.2.6"
                     /label=bla
                     /citation="[3]"
                     /citation="[4]"
                     /citation="[7]"
                     /protein_id="AAB61948.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            2062..2922
                     /gene="bla"
                     /label=bla
     sig_peptide     2062..2130
                     /gene="bla"
     mat_peptide     2131..2919
                     /gene="bla"
                     /product="beta-lactamase"
                     /EC_number="3.5.2.6"
     variation       2305
                     /gene="bla"
                     /replace="g"
                     /label=mutation of HincII site of pBR322 [3]
                     /note="mutation of HincII site of pBR322 [3]"
                     /citation="[3]"
                     /citation="[4]"
     variation       2606
                     /gene="bla"
                     /replace="c"
                     /label=mutation of PstI site of pBR322 [3]
                     /note="mutation of PstI site of pBR322 [3]"
                     /citation="[3]"
                     /citation="[4]"
     rep_origin      3082..3696
                     /standard_name="pMB1 (ColE1-like) plasmid origin of 
                     replication, as in pBR322 [4]"
                     /note="the DNA region indicated is sufficient for a 
                     functional replication origin [1]; DNA replication 
                     initiates at pos. 3681 to 3683 [4]"
                     /citation="[1]"
                     /citation="[4]"
     rep_origin      3093..3681
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     old_sequence    3240
                     /replace="c"
                     /note="according to [7], the T at position 3240 is 
                     responsible for the higher copy number of pUC derivatives 
                     [3,8], including pKSS, compared to their parent pBR322 
                     [1,4]"
                     /citation="[1]"
                     /citation="[3]"
                     /citation="[4]"
                     /citation="[7]"
                     /citation="[8]"
     CDS             3864..3956
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor fragment"
                     /label=lacI
                     /note="vestigal C-terminal portion of lac repressor; this 
                     3 portion of lacI is not functional"
                     /citation="[3]"
                     /citation="[7]"
                     /protein_id="AAB61949.1"
                     /translation="APNTQTASPRALADSLMQLARQVSRLESGQ"
     gene            3864..3956
                     /gene="lacI"
                     /label=lacI
     regulatory      3957..4061
                     /regulatory_class="promoter"
                     /standard_name="lac promoter-operator region (wild type)"
                     /note="for details and references to the lac 
                     promoter-operator region see [(in) Miller,J.H. and 
                     Reznikoff,W.S. (eds); The Operon; 2nd ed (1980); Cold 
                     Spring Harbor; pp.177-243.]"
                     /citation="[5]"
                     /citation="[7]"
     protein_bind    3957..3994
                     /standard_name="CAP binding site (wild-type sequence)"
                     /label=CAP binding site (wild-type sequence) binding site
                     /bound_moiety="catabolite activator protein (CAP)"
                     /citation="[5]"
                     /citation="[7]"
     protein_bind    3969..3990
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     gene            4005..4093
                     /gene="lacZ"
                     /label=lacZ
     promoter        4005..4035
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     regulatory      4005..4010
                     /regulatory_class="minus_35_signal"
                     /gene="lacZ"
     regulatory      4029..4034
                     /regulatory_class="minus_10_signal"
                     /gene="lacZ"
     protein_bind    4041..4061
                     /gene="lacZ"
                     /standard_name="lacO; lac operator"
                     /label=lacO binding site
                     /bound_moiety="lac repressor protein"
     protein_bind    4043..4059
                     /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)."
     primer_bind     4067..4083
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4079..4093
                     /codon_start=1
                     /gene="lacZ"
                     /product="beta-galactosidase fragment"
                     /label=lacZ
                     /note="N-terminal 5 residues of beta-galactosidase"
                     /citation="[5]"
                     /citation="[7]"
                     /citation="[9]"
                     /protein_id="AAB61950.1"
                     /translation="MTMIT"
     regulatory      4104..4123
                     /regulatory_class="promoter"
                     /standard_name="T3 promoter"
                     /citation="[5]"
                     /citation="[7]"
                     /citation="[9]"
     promoter        4104..4122
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     old_sequence    4133
                     /replace="tg"
                     /note="all pBluescript I KS (+/-) vectors (and pKSS) 
                     exhibit the deletion of the G nucleotide 5 to the KpnI 
                     site [8,9]"
                     /citation="[5]"
                     /citation="[7]"
                     /citation="[9]"