The pBIND Vector is a high-copy plasmid in which the CMV immediate early promoter drives expression of a portion of the yeast GAL4 gene (amino acids 1–147) containing a DNA-binding domain. As with the pACT Vector, the DNAbinding domain sequence is flanked by a chimeric intron, a multiple cloning region, stop codons and an SV40 late polyadenylation region (Figures 4 and 5). The fusion gene region is flanked by T7 and T3 RNA polymerase promoters for the purpose of synthesizing sense and antisense RNA products, respectively. The Renilla luciferase gene on this vector is preceded by the SV40 early promoter and a growth hormone intron. Introns can increase protein expression through mRNA stability and nuclear to cytoplasmic transport effects (9–12). A synthetic polyadenylation sequence resides 3´ of the Renilla luciferase gene. The plasmid backbone contains an f1 origin of replication for the production of ssDNA and the β-lactamase gene (Ampr) for selection in E. coli.
| 载体基本信息 | |||
| 载体名称 | pBIND | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 6360 bp | 载体类型 | Mammalian Two-Hybrid System |
| 启动子 | CMV | ||
pBIND 载体载体图谱
pBIND 载体载体序列
LOCUS Exported 6360 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6360)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..6360
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 138..517
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 518..729
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
intron 890..1022
/note="chimeric intron"
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
promoter 1067..1085
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1116..1556
/codon_start=1
/gene="Saccharomyces cerevisiae GAL4 (truncated)"
/product="DNA binding domain of the GAL4 transcriptional
activator"
/note="GAL4 DNA binding domain"
/translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
promoter complement(1640..1657)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
polyA_signal 1675..1796
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 1982..2437
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2509..2866
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2717..2852
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 3208..4143
/codon_start=1
/product="luciferase from the anthozoan coelenterate
Renilla reniformis (sea pansy)"
/note="Rluc"
/translation="MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
polyA_signal 4201..4249
/note="synthetic polyadenylation signal"
promoter 4563..4667
/gene="bla"
/note="AmpR promoter"
CDS 4668..5528
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5699..6287
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
