LOCUS       Exported               10925 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Flp expression vector pFLP3, complete sequence.
ACCESSION   AY597273
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10925)
  AUTHORS   Choi KH, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer 
            RR, Schweizer HP.
  TITLE     A Tn7-based broad-range bacterial cloning and expression system
  JOURNAL   Nat. Methods 2 (6), 443-448 (2005)
  PUBMED    15908923
REFERENCE   2  (bases 1 to 10925)
  AUTHORS   Choi KH, Gaynor J, Karkhoff-Schweizer RR, Schweizer HP.
  TITLE     Improved FRT cassettes and Flp expression vectors
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 10925)
  AUTHORS   Choi KH, Gaynor J, Karkhoff-Schweizer RR, Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (12-APR-2004) Microbiology, Immunology and Pathology, 
            Colorado State University, Fort Collins, CO 80523, USA
REFERENCE   4  (bases 1 to 10925)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10925
                     /organism="Flp expression vector pFLP3"
                     /mol_type="other DNA"
                     /db_xref="taxon:274583"
     rep_origin      194..596
                     /label=region containing ori1600
                     /note="region containing ori1600"
     rep_origin      232..583
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     CDS             597..1430
                     /codon_start=1
                     /gene="rep"
                     /product="replication protein"
                     /label=rep
                     /protein_id="AAT08993.1"
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     gene            597..1430
                     /gene="rep"
                     /label=rep
     primer_bind     1591..1607
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2338..3759)
                     /codon_start=1
                     /gene="sacB"
                     /product="levansucrase"
                     /label=sacB
                     /protein_id="AAT08994.1"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     gene            complement(2338..3759)
                     /gene="sacB"
                     /label=sacB
     promoter        complement(3760..4205)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     promoter        4420..4448
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             4496..5686
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=tet
                     /protein_id="AAT08995.1"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     gene            4496..5686
                     /gene="tet"
                     /label=tet
     CDS             complement(6054..7325)
                     /codon_start=1
                     /gene="flp"
                     /product="Flp recombinase"
                     /label=flp
                     /protein_id="AAT08996.1"
                     /translation="MPQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
                     THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
                     IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
                     SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
                     KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
                     SYNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
                     TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
                     YPAWNGIISQEVLDYLSSYINRRI"
     gene            complement(6054..7325)
                     /gene="flp"
                     /label=flp
     RBS             7329..7337
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3 end of the 16S rRNA (Chen et al., 1994)"
     CDS             7426..8139
                     /codon_start=1
                     /gene="cI"
                     /allele="cI-857"
                     /product="lambda repressor"
                     /label=cI
                     /protein_id="AAT08997.1"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     gene            7426..8139
                     /gene="cI"
                     /allele="cI-857"
                     /label=cI
     primer_bind     complement(8428..8444)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8452..8468
                     /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(8476..8506)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8521..8542
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    8780..9036
                     /note="oriT; contains origin of transfer"
     oriT            8926..9034
                     /note="incP origin of transfer"
     rep_origin      complement(9106..9694)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(9865..10725)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=bla
                     /protein_id="AAT08998.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     gene            complement(9865..10725)
                     /gene="bla"
                     /label=bla
     promoter        complement(10726..10830)
                     /gene="bla"
                     /label=AmpR promoter