LOCUS       Exported                6872 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Temperature-sensitive shuttle vector pFNLTP9, complete sequence.
ACCESSION   AY622905
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6872)
  AUTHORS   Maier TM, Havig A, Casey M, Nano FE, Frank DW, Zahrt TC.
  TITLE     Construction and characterization of a highly efficient Francisella 
            shuttle plasmid
  JOURNAL   Appl. Environ. Microbiol. 70 (12), 7511-7519 (2004)
  PUBMED    15574954
REFERENCE   2  (bases 1 to 6872)
  AUTHORS   Maier TM, Havig A, Casey M, Nano FE, Frank DW, Zahrt TC.
  TITLE     Direct Submission
  JOURNAL   Submitted (07-MAY-2004) Microbiology and Molecular Genetics, Medical
            College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 
            53226, USA
REFERENCE   3  (bases 1 to 6872)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6872
                     /organism="Temperature-sensitive shuttle vector pFNLTP9"
                     /mol_type="other DNA"
                     /label=vector for Francisella spp. and Escherichia coli
                     /note="vector for Francisella spp. and Escherichia coli"
                     /db_xref="taxon:280786"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    234..258
                     /note="multiple cloning site; MCS; derived from 
                     pCR2.1-TOPO"
     mRNA            527..1420
                     /product="unknown"
                     /label=unknown mRNA
     CDS             542..1420
                     /codon_start=1
                     /product="unknown"
                     /label=unknown
                     /note="orf3"
                     /protein_id="AAT44340.1"
                     /translation="MKDKAAKNRDFRKTILSVLQRNKDGSFATQANRKSILLQATKDLK
                     KVGFSKVTAENFGNKHCYALRDHWRAQGLATATIKNRLACLRWLGEKMGKELPDNRKLE
                     IENRKYSDNSINKAQEIDFKAISALTDRQALAIQLQREFGLRREESLKFQPSYAIKEHK
                     IELKSSWTKGGRPREIPILNEKQRELLEKVKEVAGKGSLIESEKSYKQAMEHFTTRCQR
                     AGIKNVHGFRHAYAQDRYRQLTGRECPKNGGLTSKQLTPEQKQQDYEARMTISEELGHG
                     REDVTVNYLGR"
     CDS             complement(1637..2059)
                     /codon_start=1
                     /product="RepB protein"
                     /label=RepB protein
                     /note="orf2"
                     /protein_id="AAT44341.1"
                     /translation="MNIEIFKFSKKHKKINKKLIDLTKVKQVVTTGYSFIVVYQRGRKI
                     ELSYESSYWHAKKDLDNSNSDVGITYSYIKKDKRGPCHKDNYLRDSKGRLLKDWGKIEE
                     LHEKYPKIGKIWYISREETDKLLKDFTYKNGNLIMV"
     gene            complement(2062..3139)
                     /gene="repA"
                     /label=repA
     mRNA            complement(2062..3104)
                     /gene="repA"
                     /product="mutant RepA protein"
                     /label=repA mRNA
     CDS             complement(2062..3081)
                     /codon_start=1
                     /gene="repA"
                     /product="mutant RepA protein"
                     /label=repA
                     /note="orf1; RepA M120I"
                     /protein_id="AAT44342.1"
                     /translation="MKEVKNVNAGKEIAMSNTLVAGKYSLTKEEQNLIFLVASMVKRED
                     KEFHRYKISLSDLEKATGVKHNRVRLKQLMHSIMSKPVWLNKEQTKIANWFAYIEADPK
                     SSALICEFHWSLMPHIIQLQEYFTKAERQLLFSFKSKYSSRLYLLLKSKLGEQAGYANI
                     VDCVLYVDDMINDFDLPKSYSNRYSNFKNKFLLPALEEINQLSDIFVTYDDKDHHRKHG
                     RKITNIKFTVSKVAETEEEFKQKLLDTKNKEDYIPIEASERLREVVLSDELDLDLYYVR
                     SIFRHHHLHDIERVCDDTWRNWDNKKIKDRKRVFIANIKKLEKKPEENHKLPFGFEEV"
     variation       2722
                     /gene="repA"
                     /replace="c"
                     /note="compared to pFNLTP1 wild-type repA in GenBank 
                     Accession Number AY622904; results in isoleucine to 
                     methionine amino acid change"
     regulatory      complement(3111..3116)
                     /regulatory_class="minus_10_signal"
                     /gene="repA"
     regulatory      complement(3134..3139)
                     /regulatory_class="minus_35_signal"
                     /gene="repA"
     repeat_region   3168..3262
     repeat_region   3317..3347
     CDS             3452..3637
                     /codon_start=1
                     /product="unknown"
                     /label=unknown
                     /note="orf5; hybrid ORF; contains partial phd sequence"
                     /protein_id="AAT44343.1"
                     /translation="MQTVNYSTFRNELSDSMDRVTKNHSPMIVTRGSKKEAVVMMSLED
                     SSLPFSPRSPAFPVKL"
     CDS             4260..5054
                     /codon_start=1
                     /gene="npt"
                     /product="kanamycin resistance protein"
                     /label=npt
                     /note="neomycin phosphotransferase"
                     /protein_id="AAT44344.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            4260..5054
                     /gene="npt"
                     /label=npt
     CDS             5072..5932
                     /codon_start=1
                     /gene="bla"
                     /product="ampicillin resistance protein"
                     /label=bla
                     /note="beta-lactamase"
                     /protein_id="AAT44345.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            5072..5932
                     /gene="bla"
                     /label=bla
     rep_origin      6077..6750
                     /label=pUC origin derived from pCR2.1-TOPO
                     /note="pUC origin derived from pCR2.1-TOPO"
     rep_origin      6103..6691
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"