LOCUS       Exported               12925 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pBiFCt-2in1-NN, complete sequence.
ACCESSION   KT600154
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12925)
  AUTHORS   Grefen C, Blatt MR.
  TITLE     A 2in1 cloning system enables ratiometric bimolecular fluorescence 
            complementation (rBiFC)
  JOURNAL   BioTechniques 53 (5), 311-314 (2012)
  PUBMED    23066669
REFERENCE   2  (bases 1 to 12925)
  AUTHORS   Grefen C.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-AUG-2015) ZMBP Developmental Genetics, University of 
            Tuebingen, Auf der Morgenstelle 32, Tuebingen 72076, Germany
REFERENCE   3  (bases 1 to 12925)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..12925
                     /organism="Cloning vector pBiFCt-2in1-NN"
                     /mol_type="other DNA"
                     /db_xref="taxon:1792577"
     regulatory      16..518
                     /regulatory_class="promoter"
                     /note="CaMV35S plus Omega enhancer"
     promoter        93..437
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             531..995
                     /codon_start=1
                     /product="nYFP (aa1-154)"
                     /label=nYFP
                     /protein_id="AMD02805.1"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA"
     CDS             1026..1053
                     /codon_start=1
                     /product="HA-tag"
                     /label=HA-tag
                     /protein_id="AMD02806.1"
                     /translation="MYPYDVPDY"
     CDS             1029..1055
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_recomb     1065..1189
                     /label=attR3 gateway recombination site
                     /note="attR3 gateway recombination site"
     protein_bind    1066..1189
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      1200..1297
                     /regulatory_class="promoter"
                     /note="PlacZ"
     promoter        1224..1254
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1262..1278
                     /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     1286..1302
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1298..1516
                     /codon_start=1
                     /product="lacZ"
                     /label=lacZ
                     /protein_id="AMD02807.1"
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRS"
     misc_recomb     complement(1563..1687)
                     /label=attR2 gateway recombination site
                     /note="attR2 gateway recombination site"
     protein_bind    complement(1563..1687)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      1719..1924
                     /regulatory_class="terminator"
                     /note="T35S terminator"
     regulatory      1937..2439
                     /regulatory_class="promoter"
                     /note="CaMV35S plus Omega enhancer"
     promoter        2014..2358
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             2446..3120
                     /codon_start=1
                     /product="mRFP1 red fluorescent protein"
                     /label=mRFP1 red fluorescent protein
                     /protein_id="AMD02808.1"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     regulatory      3177..3385
                     /regulatory_class="terminator"
                     /note="T35S terminator"
     regulatory      3398..3900
                     /regulatory_class="promoter"
                     /note="CaMV35S plus Omega enhancer"
     promoter        3475..3819
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             3907..4161
                     /codon_start=1
                     /product="cYFP (aa155-240)"
                     /label=cYFP
                     /protein_id="AMD02809.1"
                     /translation="MDKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDN
                     HYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     CDS             4195..4227
                     /codon_start=1
                     /product="MYC-tag"
                     /label=MYC-tag
                     /protein_id="AMD02810.1"
                     /translation="MEQKLISEEDL"
     CDS             4198..4227
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_recomb     4246..4370
                     /label=attR1 gateway recombination site
                     /note="attR1 gateway recombination site"
     protein_bind    4246..4370
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4395..4425
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             4479..5138
                     /codon_start=1
                     /product="CmR chloramphenicol acetyl transferase"
                     /label=CmR chloramphenicol acetyl transferase
                     /protein_id="AMD02811.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             5480..5785
                     /codon_start=1
                     /product="ccdB gyrase inhibitor"
                     /label=ccdB gyrase inhibitor
                     /protein_id="AMD02812.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     misc_recomb     complement(5826..5950)
                     /label=attR4 gateway recombination site
                     /note="attR4 gateway recombination site"
     protein_bind    complement(5826..5950)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      5982..6187
                     /regulatory_class="terminator"
                     /note="T35S terminator"
     misc_feature    complement(6208..6537)
                     /label=LB T-DNA left border
                     /note="LB T-DNA left border"
     misc_feature    6275..6299
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     misc_feature    6543..7787
                     /product="SpecR aminoglycoside resistance protein"
     CDS             6820..7611
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      7857..8445
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      8061..8741
                     /label=pBR322 ori replication in Escherichia coli
                     /note="pBR322 ori replication in Escherichia coli"
     misc_feature    8631..8771
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      9115..9309
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     rep_origin      9151..11745
                     /label=pVS1 ori replication in Agrobacterium tumefaciens
                     /note="pVS1 ori replication in Agrobacterium tumefaciens"
     CDS             complement(9375..10448)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     CDS             complement(10877..11506)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     misc_feature    12722..12917
                     /label=RB T-DNA right border
                     /note="RB T-DNA right border"
     misc_feature    12837..12861
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"