LOCUS       Exported                7317 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pZD_2-3-T14+T14x3+72-mKate2, complete sequence.
ACCESSION   KP408214
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7317)
  AUTHORS   Li Y, Jiang Y, Chen H, Liao W, Li Z, Weiss R, Xie Z.
  TITLE     Modular construction of mammalian gene circuits using TALE 
            transcriptional repressors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7317)
  AUTHORS   Li Y, Jiang Y, Chen H, Liao W, Li Z, Weiss R, Xie Z.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-JAN-2015) Bioinformatics Division/Center for Synthetic
            & Systems Biology, Tsinghua National Laboratory for Information 
            Science and Technology, Tsinghua University, Beijing, Beijing 
            100084, China
REFERENCE   3  (bases 1 to 7317)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7317
                     /organism="Cloning vector pZD_2-3-T14+T14x3+72-mKate2"
                     /mol_type="other DNA"
                     /db_xref="taxon:1602995"
     CDS             complement(1..861)
                     /codon_start=1
                     /product="AmpR"
                     /label=AmpR
                     /protein_id="AJF17161.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(862..966)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      992..1447
                     /label=f1 origin
                     /note="f1 origin"
     primer_bind     1589..1605
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1615..1633
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1648..2451
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    2530..2555
                     /label=SA
                     /note="splice acceptor site"
     protein_bind    3220..3240
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    3288..3382
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     misc_feature    3288..3304
                     /label=UAS
                     /note="UAS"
     misc_feature    3307..3323
                     /label=UAS
                     /note="UAS"
     misc_feature    3326..3342
                     /label=UAS
                     /note="UAS"
     misc_feature    3345..3361
                     /label=UAS
                     /note="UAS"
     misc_feature    3364..3380
                     /label=UAS
                     /note="UAS"
     misc_feature    3396..3412
                     /label=TALER14 bs
                     /note="TALER14 bs"
     misc_feature    3419..3478
                     /label=miniCMV
                     /note="miniCMV"
     misc_feature    3485..3501
                     /label=TALER14 bs
                     /note="TALER14 bs"
     misc_feature    3502..3518
                     /label=TALER14 bs
                     /note="TALER14 bs"
     misc_feature    3519..3535
                     /label=TALER14 bs
                     /note="TALER14 bs"
     protein_bind    3551..3575
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3611..4339
                     /codon_start=1
                     /product="6xHis-mKate2"
                     /label=6xHis-mKate2
                     /protein_id="AJF17162.1"
                     /translation="MHHHHHHMVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEG
                     TQTMRIKAVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTY
                     EDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLE
                     GRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQH
                     EVAVARYCDLPSKLGHKLNS"
     CDS             3614..3631
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3632..4324
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGH"
     protein_bind    complement(4370..4394)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_site      4549..4604
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     primer_bind     complement(4965..4981)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4989..5005
                     /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(5013..5043)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5058..5079
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    5252..6088
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        complement(6118..6136)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6157..6173)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6181..6197
                     /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(6205..6235)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6250..6271
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(6544..7163)
                     /direction=LEFT
                     /label=pBR322 origin
                     /note="pBR322 origin"
     rep_origin      complement(6559..7147)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"