Synthetic Gene DataBase
 

Synthetic Gene 257


 
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Field NameNatural GeneSynthetic Gene
SGDB Gene ID228257
GenBank AccessionV00296AY192356
GenBank GI4190137780056
Gene NameLacZLacZco
Gene Length (bp)30693069
SpeciesE. coliMus musculus; Homo sapiens; Chinese Hamster
StrainsEC 3.2.1.23NIH3T3; HeLa, A549; CHO-K1
CDSatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgtt
acccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagag
gcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcc
tggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggcc
gatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctacacc
aacgtaacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggt
tgttactcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaatt
atttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttac
ggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaa
aaccgcctcgcggtgatggtgctgcgttggagtgacggcagttatctggaagatcaggat
atgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaa
atcagcgatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggag
gctgaagttcagatgtgcggcgagttgcgtgactacctacgggtaacagtttctttatgg
cagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgag
cgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtgg
agcgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacg
ctgattgaagcagaagcctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctg
ctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatcct
ctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcag
aacaactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctg
tgcgaccgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtg
ccaatgaatcgtctgaccgatgatccgcgctggctaccggcgatgagcgaacgcgtaacg
cgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaatgaa
tcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcc
cgcccggtgcagtatgaaggcggcggagccgacaccacggccaccgatattatttgcccg
atgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaa
aaatggctttcgctacctggagagacgcgcccgctgatcctttgcgaatacgcccacgcg
atgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatccccgt
ttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaac
ggcaacccgtggtcggcttacggcggtgattttggcgatacgccgaacgatcgccagttc
tgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaa
caccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaa
tacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaag
ccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaa
ctgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtg
caaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgt
ctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgacc
accagcgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgccag
tcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgc
gatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcatt
gaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcg
ttgttgcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcg
tggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagt
ggtcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcgg
attggcctgaactgccagctggcgcaggtagcagagcgggtaaactggctcggattaggg
ccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgcca
ttgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacg
cgcgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgc
tacagtcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggc
acatggctgaatatcgacggtttccatatggggattggtggcgacgactcctggagcccg
tcagtatcggcggaattccagctgagcgccggtcgctaccattaccagttggtctggtgt
caaaaataa
atgatcaccgactccctggccgtggtgctgcagcgccgcgactgggagaaccccggcgtg
acccagctgaaccgcctggccgcccacccccccttcgcctcctggcgcaactccgaggag
gcccgcaccgaccgcccctcccagcagctgcgctccctgaacggcgagtggcgcttcgcc
tggttccccgcccccgaggccgtgcccgagtcctggctggagtgcgacctgcccgaggcc
gacaccgtggtggtgccctccaactggcagatgcacggctacgacgcccccatctacacc
aacgtgacctaccccatcaccgtgaacccccccttcgtgcccaccgagaaccccaccggc
tgctactccctgaccttcaacgtggacgagtcctggctgcaggagggccagacccgcatc
atcttcgacggcgtgaactccgccttccacctgtggtgcaacggccgctgggtgggctac
ggccaggactcccgcctgccctccgagttcgacctgtccgccttcctgcgcgccggcgag
aaccgcctggccgtgatggtgctgcgctggtccgacggctcctacctggaggaccaggac
atgtggcgcatgtccggcatcttccgcgacgtgtccctgctgcacaagcccaccacccag
atctccgacttccacgtggccacccgcttcaacgacgacttctcccgcgccgtgctggag
gccgaggtgcagatgtgcggcgagctgcgcgactacctgcgcgtgaccgtgtccctgtgg
cagggcgagacccaggtggcctccggcaccgcccccttcggcggcgagatcatcgacgag
cgcggcggctacgccgaccgcgtgaccctgcgcctgaacgtggagaaccccaagctgtgg
tccgccgagatccccaacctgtaccgcgccgtggtggagctgcacaccgccgacggcacc
ctgatcgaggccgaggcctgcgacgtgggcttccgcgaggtgcgcatcgagaacggcctg
ctgctgctgaacggcaagcccctgctgatccgcggcgtgaaccgccacgagcaccacccc
ctgcacggccaggtgatggacgagcagaccatggtgcaggacatcctgctgatgaagcag
aacaacttcaacgccgtgcgctgctcccactaccccaaccaccccctgtggtacaccctg
tgcgaccgctacggcctgtacgtggtggacgaggccaacatcgagacccacggcatggtg
cccatgaaccgcctgaccgacgacccccgctggctgcccgccatgtccgagcgcgtgacc
cgcatggtgcagcgcgaccgcaaccacccctccgtgatcatctggtccctgggcaacgag
tccggccacggcgccaaccacgacgccctgtaccgctggatcaagtccgtggacccctcc
cgccccgtgcagtacgagggcggcggcgccgacaccaccgccaccgacatcatctgcccc
atgtacgcccgcgtggacgaggaccagcccttccccgccgtgcccaagtggtccatcaag
aagtggctgtccctgcccggcgagacccgccccctgatcctgtgcgagtacgcccacgcc
atgggcaactccctgggcggcttcgccaagtactggcaggccttccgccagtacccccgc
ctgcagggcggcttcgtgtgggactgggtggaccagtccctgatcaagtacgacgagaac
ggcaacccctggtccgcctacggcggcgacttcggcgacacccccaacgaccgccagttc
tgcatgaacggcctggtgttcgccgaccgcaccccccaccccgccctgaccgaggccaag
caccagcagcagttcttccagttccgcctgtccggccagaccatcgaggtgacctccgag
tacctgttccgccactccgacaacgagctgctgcactggatggtggccctggacggcaag
cccctggcctccggcgaggtgcccctggacgtggccccccagggcaagcagctgatcgag
ctgcccgagctgccccagcccgagtccgccggccagctgtggctgaccgtgcgcgtggtg
cagcccaacgccaccgcctggtccgaggccggccacatctccgcctggcagcagtggcgc
ctggccgagaacctgtccgtgaccctgcccgccgcctcccacgccatcccccacctgacc
acctccgagatggacttctgcatcgagctgggcaacaagcgctggcagttcaaccgccag
tccggcttcctgtcccagatgtggatcggcgacaagaagcagctgctgacccccctgcgc
gaccagttcacccgcgcccccctggacaacgacatcggcgtgtccgaggccacccgcatc
gaccccaacgcctgggtggagcgctggaaggccgccggccactaccaggccgaggccgcc
ctgctgcagtgcaccgccgacaccctggccgacgccgtgctgatcaccaccgcccacgcc
tggcagcaccagggcaagaccctgttcatctcccgcaagacctaccgcatcgacggctcc
ggccagatggccatcaccgtggacgtggaggtggcctccgacaccccccaccccgcccgc
atcggcctgaactgccagctggcccaggtggccgagcgcgtgaactggctgggcctgggc
ccccaggagaactaccccgaccgcctgaccgccgcctgcttcgaccgctgggacctgccc
ctgtccgacatgtacaccccctacgtgttcccctccgagaacggcctgcgctgcggcacc
cgcgagctgaactacggcccccaccagtggcgcggcgacttccagttcaacatctcccgc
tactcccagcagcagctgatggagacctcccaccgccacctgctgcacgccgaggagggc
acctggctgaacatcgacggcttccacatgggcatcggcggcgacgactcctggtccccc
tccgtgtccgccgagttccagctgtccgccggccgctaccactaccagctggtgtggtgc
cagaagtag
5' End
3' End
Notes
Expression VectorpcDNA3.1pcDNA3.1
Assay MethodsX-gal staining, Northern BlotX-gal staining, Northern Blot
Results1.89+/-0.15 ng Beta-gal per mg total cell proteinmRNA levels were approximately twice as high as wild-type LacZ. The resulting protein expression was approximately 15 fold higher than the wild-type gene as estimated from X-gal staining (28.6+/-0.5 ng Beta-gal per mg total cell protein).
Protein Functionreporter gene
Recoding PurposeTo improve expression
Synthesized ByAuthors
Recoding MethodThe Beta-gal protein sequence was back-translated using optimal codon usage for high level mammalian
gene expression. A Kozak translational initiation consensus sequence was placed in front of the
coding sequence.
Publication Author(s)Anson DS, Limberis M.
Corresponding AuthorDonald S. Anson
Corresponding AddressDepartment of Chemical Pathology, Women's and Children's Hospital, 72 King William Road, North Adelaide, SA 5006, Australia.
Publication Year2004
Publication TitleAn improved beta-galactosidase reporter gene.
AbstractThe coding sequence for the E. coli beta-galactosidase gene was codon-optimised for expression in mammalian cells. When expressed in mammalian cells the codon-optimised gene results in the expression of beta-galactosidase at levels 15-fold higher than those resulting from an analogous construct containing the native E. coli gene sequence. RNA analysis suggests the enhancement of beta-galactosidase expression is due both to enhanced transcript stability and increased translational efficiency. When used in a lentiviral construct the codon-optimised gene results in an approximately five-fold increase in apparent titre, as determined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining, in comparison to an analogous construct containing the native E. coli gene. Southern blot analysis shows this is due to an increased efficiency of detection of transduced cells. In addition, codon-optimisation results in the elimination of several cryptic splice acceptor sites that are present in the native E. coli gene sequence. In a lentiviral vector containing a 5' splice donor the use of the codon-optimised gene in place of the native E. coli beta-galactosidase gene resulted in increased amounts of un-spliced, full-length genomic RNA. Therefore, as a marker/reporter gene in mammalian cells the codon-optimised beta-galactosidase gene has a number of advantages over the native E. coli gene sequence. A variant of the codon-optimised beta-galactosidase gene sequence that includes an effective nuclear localisation signal was also made.
JournalJ Biotechnol.. 108(1): 17-30.
SummaryThe authors wished to produce an improved Beta-gal reporter for use in mammalian cells. Wild-type Beta-gal activity was disappointingly low and required prolonged incubation with X-gal. The authors approached the problem with by performing codon optimization on the LacZ gene. The Beta-gal protein sequence was back-translated using optimal codon usage for high level mammalian gene expression. A Kozak translational initiation consensus sequence was placed in front of the coding sequence. Northern blot analysis showed that higher mRNA resulted from recoded LacZ. Ultimately, codon optimization improved Beta-galactosidase expression by 15 fold, as a consequence of both enhanced mRNA stability and translational efficiency. The authors also produced protein 2 variants that localized to the nucleus successfully.
Comments
Discussion
PubMed ID14741766
Submitter NameZheng, Yuanpu
Submitter AddressUMBC
Entry ConfirmationNo
 
 

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