LOCUS       Exported                8730 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pBOB, complete sequence.
ACCESSION   JF927991
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8730)
  AUTHORS   Wachsman G, Heidstra R, Scheres B.
  TITLE     Distinct Cell-Autonomous Functions of RETINOBLASTOMA-RELATED in 
            Arabidopsis Stem Cells Revealed by the Brother of Brainbow Clonal 
            Analysis System
  JOURNAL   Plant Cell 23 (7), 2581-2591 (2011)
  PUBMED    21742994
REFERENCE   2  (bases 1 to 8730)
  AUTHORS   Wachsman G, Heidstra R, Scheres B.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-MAY-2011) Molecular Genetics, Utrecht University, 
            Padualaan 8, Utrecht 3584CH, The Netherlands
REFERENCE   3  (bases 1 to 8730)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8730
                     /organism="Cloning vector pBOB"
                     /mol_type="other DNA"
                     /label=based on pGreenII
                     /note="based on pGreenII"
                     /db_xref="taxon:1042257"
     CDS             320..1135
                     /codon_start=1
                     /gene="aph(3)-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANVVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      1306..1894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    1985..2009
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     protein_bind    2248..2269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2284..2314
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2322..2338
                     /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     2346..2362
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2383..2401
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      2428..2829
                     /regulatory_class="promoter"
                     /note="35S"
     promoter        2485..2830
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     misc_recomb     2830..2863
                     /label=lox2272
                     /note="lox2272"
     protein_bind    2830..2863
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
     misc_recomb     2870..2903
                     /label=loxN
                     /note="loxN"
     protein_bind    2870..2903
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GTATACCT). loxN sites are compatible with each 
                     other, but incompatible with loxP or lox2272 sites."
     CDS             2904..3644
                     /codon_start=1
                     /product="venus YFP"
                     /label=venus YFP
                     /note="SV40 nuclear-localized"
                     /protein_id="AEH43767.1"
                     /translation="MPKKKRKVVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDA
                     TYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQER
                     TIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITAD
                     KQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEK
                     RDHMVLLEFVTAAGITLGMDELYK"
     CDS             2907..2927
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2928..3644
                     /codon_start=1
                     /product="Venus YFP with monomerizing A206K mutation (Nagai
                     et al., 2002)"
                     /label=mVenus
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     regulatory      3645..4136
                     /regulatory_class="terminator"
                     /note="3A terminator"
     misc_recomb     4169..4202
                     /label=lox2272
                     /note="lox2272"
     protein_bind    4169..4202
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites."
     CDS             4203..5003
                     /codon_start=1
                     /product="Cypet"
                     /label=Cypet
                     /note="ER-localized"
                     /protein_id="AEH43768.1"
                     /translation="MKTNLFLFLIFSLLLSLSSAEFGAVSKGEELFGGIVPILVELEGD
                     VNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQ
                     HDFFKSVMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH
                     KLEYNYISHNVYITADKQKNGIKANFKARHNITDGSVQLADHYQQNTPIGDGPVILPDN
                     HYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKHDEL"
     CDS             4275..4988
                     /codon_start=1
                     /product="cyan fluorescent protein optimized for FRET with 
                     YPet (Nguyen and Daugherty, 2005)"
                     /label=CyPet
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFGGIVPILVELEGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSVMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIKA
                     NFKARHNITDGSVQLADHYQQNTPIGDGPVILPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     regulatory      5004..5289
                     /regulatory_class="terminator"
                     /note="nos terminator"
     terminator      5014..5266
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     misc_recomb     5296..5329
                     /label=loxN
                     /note="loxN"
     protein_bind    5296..5329
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GTATACCT). loxN sites are compatible with each 
                     other, but incompatible with loxP or lox2272 sites."
     CDS             5330..6106
                     /codon_start=1
                     /product="TagRFP"
                     /label=TagRFP
                     /note="ER-localized"
                     /protein_id="AEH43769.1"
                     /translation="MKTNLFLFLIFSLLLSLSSAEFTGSELIKENMHMKLYMEGTVNNH
                     HFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFK
                     QSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGW
                     EANTEMLYPADGGLEGRSDMALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRL
                     ERIKEADKETYVEQHEVAVARYCDLPSKLGHKHDEL"
     regulatory      6107..6363
                     /regulatory_class="terminator"
                     /note="35 terminator"
     polyA_signal    6184..6360
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        complement(6384..6402)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6412..6428)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6646..6991
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             7024..7587
                     /codon_start=1
                     /gene="DHFR"
                     /product="methotrexate resistance protein"
                     /label=DHFR
                     /note="confers resistance to methotrexate in planta"
                     /protein_id="AEH43770.1"
                     /translation="MVRPLNCIVAVSQNMGIGKNGDRPWPPLRNEFKYFQRMTTTSSVE
                     GKQNLVIMGRKTWFSIPEKNRPLKDRINIVLSRELKEPPRGAHFLAKSLDDALRLIEQP
                     ELASKVDMVWIVGGSSVYQEAMNQPGHLRLFVTRIMQEFESDTFFPEIDLGKYKLLPEY
                     PGVLSEVQEEKGIKYKFEVYEKKD"
     gene            7024..7587
                     /gene="DHFR"
                     /label=DHFR
     polyA_signal    7983..8159
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     misc_feature    8167..8189
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     rep_origin      8324..29
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"