Primers

for
UG-07
Green
Fluorescent
Protein (GFP)
Marrium Mansoor

Assignment # 1

Primers for Green Fluorescent

Protein (GFP)
Primer
Primers are short DNA/RNA sequences that are complementary to longer, original
DNA sequences and are needed for DNA replication (or amplification) as the DNAsynthesizing enzyme DNA polymerase can only extend an existing nucleotide
sequence and not start one from scratch. They are used synthetically in PCR.
To select primers for gene of Green Fluorescent Protein found in Aequorea victoria,
we follow these steps:
1. Find nucleotide sequence of GFP from NCBI. (http://www.ncbi.nlm.nih.gov )
2. Copy FASTA of sequence into primer generating software (e.g.
http://bioinfo.ut.ee/primer3/)
3. Set specifications for primer sequences
4. Pick primers.
5. Confirm presence of primer sequences within the target sequence.

Nucleotide sequence of Green Fluorescent Protein

ORIGIN
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961

tacacacgaa
cccaattctt
gggtgaaggt
actacctgtt
aagataccca
tgtacaggaa
caagtttgaa
agatggaaac
catggcagac
agatggaagc
tgtcctttta
cgaaaagaga
catggatgaa
attactaaaa
agattcatta
taaacaggct
ctcaaa

taaaagataa
gttgaattag
gatgcaacat
ccatggccaa
gatcatatga
agaactatat
ggtgataccc
attcttggac
aaacaaaaga
gttcaactag
ccagacaacc
gaccacatgg
ctatacaaat
tctcagggtt
aaattgtatg
acttggagtg

caaagatgag
atggtgatgt
acggaaaact
cacttgtcac
aacagcatga
ttttcaaaga
ttgttaatag
acaaattgga
atggaatcaa
cagaccatta
attacctgtc
tccttcttga
aaatgtccag
cctggttaaa
aataatttat
tattcttaat

taaaggagaa
taatgggcac
tacccttaaa
tactttctct
ctttttcaag
tgacgggaac
aatcgagtta
atacaactat
agttaacttc
tcaacaaaat
cacacaatct
gtttgtaaca
acttccaatt
ttcaggctga
tgatgttatt
tctatattaa

gaacttttca
aaattttctg
tttatttgca
tatggtgttc
agtgccatgc
tacaagacac
aaaggtattg
aactcacaca
aaaattagac
actccaattg
gccctttcga
gctgctggga
gacactaaag
gatattattt
gatagaggtt
ttacaatttg

ctggagttgt
tcagtggaga
ctactggaaa
aatgcttttc
ccgaaggtta
gtgctgaagt
attttaaaga
atgtatacat
acaacattga
gcgatggccc
aagatcccaa
ttacacatgg
tgtccgaaca
atatatttat
attttcttat
atttgacttg

Source:
Aequorea victoria green-fluorescent protein mRNA, complete cds
GenBank: M62653.1

(Aequorea victoria green-fluorescent protein mRNA, complete cds, n.d.)

Possible Primers
No mispriming library specified
Using 1-based sequence positions
OLIGO
start len
tm
LEFT PRIMER
110
20
59.68
RIGHT PRIMER
304
20
60.71
SEQUENCE SIZE: 966
INCLUDED REGION SIZE: 966

gc%
any
3' seq
60.00 3.00 0.00 GTCAGTGGAGAGGGTGAAGG
50.00 4.00 2.00 TACATAACCTTCGGGCATGG

PRODUCT SIZE: 195, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 0.00
1 TACACACGAATAAAAGATAACAAAGATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGT
61 CCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGA
>>>>>>>>>>>
121 GGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAA
>>>>>>>>>
181 ACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTC
241 AAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTA
<<<<<<<<<<<<<<<<
301 TGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGT
<<<<
361 CAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGA
421 AGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACAT
481 CATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGA
541 AGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCC
601 TGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAA
661 CGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGG
721 CATGGATGAACTATACAAATAAATGTCCAGACTTCCAATTGACACTAAAGTGTCCGAACA
781 ATTACTAAAATCTCAGGGTTCCTGGTTAAATTCAGGCTGAGATATTATTTATATATTTAT

841 AGATTCATTAAAATTGTATGAATAATTTATTGATGTTATTGATAGAGGTTATTTTCTTAT
901 TAAACAGGCTACTTGGAGTGTATTCTTAATTCTATATTAATTACAATTTGATTTGACTTG
961 CTCAAA
KEYS (in order of precedence):
>>>>>> left primer
<<<<<< right primer
ADDITIONAL OLIGOS
start

len

tm

gc%

any

3' seq

1 LEFT PRIMER
113
20
59.68
55.00 1.00 1.00 AGTGGAGAGGGTGAAGGTGA
RIGHT PRIMER
304
20
60.71
50.00 4.00 2.00 TACATAACCTTCGGGCATGG
PRODUCT SIZE: 192, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 1.00
2 LEFT PRIMER
111
20
59.68
55.00 3.00 0.00 TCAGTGGAGAGGGTGAAGGT
RIGHT PRIMER
304
20
60.71
50.00 4.00 2.00 TACATAACCTTCGGGCATGG
PRODUCT SIZE: 194, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 1.00
3 LEFT PRIMER
628
20
59.97
50.00 3.00 0.00 GTCCACACAATCTGCCCTTT
RIGHT PRIMER
817
21
59.83
47.62 5.00 2.00 GCCTGAATTTAACCAGGAACC
PRODUCT SIZE: 190, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00
4 LEFT PRIMER
128
20
59.40
45.00 6.00 0.00 GGTGATGCAACATACGGAAA
RIGHT PRIMER
304
20
60.71
50.00 4.00 2.00 TACATAACCTTCGGGCATGG
PRODUCT SIZE: 177, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00

Statistics
con
too
in
in
no
tm
tm high high
sid many
tar excl
bad
GC
too
too
any
3'
ered
Ns
get
reg
GC% clamp
low high compl compl
Left
8213
0
0
0
565
0 3939 1496
0
6
Right
8264
0
0
0
786
0 3931 1442
2
2
Pair Stats:
considered 1990, unacceptable product size 1977, ok 13

primer3 release 1.1.4

(primer3_results.cgi release 0.4.0)

(htt4)

poly
X
0
0

high
end
stab
70
66

ok
2137
2035

Primer Sequences in GFP

Primer pair 1
Forward primer: GTCAGTGGAGAGGGTGAAGG
Reverse primer: TACATAACCTTCGGGCATGG
Sequence start point: 110 and 304

1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961

tacacacgaa
cccaattctt
gggtgaaggt
actacctgtt
aagataccca
tgtacaggaa
caagtttgaa
agatggaaac
catggcagac
agatggaagc
tgtcctttta
cgaaaagaga
catggatgaa
attactaaaa
agattcatta
taaacaggct
ctcaaa

taaaagataa
gttgaattag
gatgcaacat
ccatggccaa
gatcatatga
agaactatat
ggtgataccc
attcttggac
aaacaaaaga
gttcaactag
ccagacaacc
gaccacatgg
ctatacaaat
tctcagggtt
aaattgtatg
acttggagtg

caaagatgag
atggtgatgt
acggaaaact
cacttgtcac
aacagcatga
ttttcaaaga
ttgttaatag
acaaattgga
atggaatcaa
cagaccatta
attacctgtc
tccttcttga
aaatgtccag
cctggttaaa
aataatttat
tattcttaat

taaaggagaa
taatgggcac
tacccttaaa
tactttctct
ctttttcaag
tgacgggaac
aatcgagtta
atacaactat
agttaacttc
tcaacaaaat
cacacaatct
gtttgtaaca
acttccaatt
ttcaggctga
tgatgttatt
tctatattaa

gaacttttca
aaattttctg
tttatttgca
tatggtgttc
agtgccatgc
tacaagacac
aaaggtattg
aactcacaca
aaaattagac
actccaattg
gccctttcga
gctgctggga
gacactaaag
gatattattt
gatagaggtt
ttacaatttg

ctggagttgt
tcagtggaga
ctactggaaa
aatgcttttc
ccgaaggtta
gtgctgaagt
attttaaaga
atgtatacat
acaacattga
gcgatggccc
aagatcccaa
ttacacatgg
tgtccgaaca
atatatttat
attttcttat
atttgacttg

taaaggagaa
taatgggcac
tacccttaaa
tactttctct
ctttttcaag
tgacgggaac
aatcgagtta
atacaactat
agttaacttc
tcaacaaaat
cacacaatct
gtttgtaaca
acttccaatt
ttcaggctga

gaacttttca
aaattttctg
tttatttgca
tatggtgttc
agtgccatgc
tacaagacac
aaaggtattg
aactcacaca
aaaattagac
actccaattg
gccctttcga
gctgctggga
gacactaaag
gatattattt

ctggagttgt
tcagtggaga
ctactggaaa
aatgcttttc
ccgaaggtta
gtgctgaagt
attttaaaga
atgtatacat
acaacattga
gcgatggccc
aagatcccaa
ttacacatgg
tgtccgaaca
atatatttat

Primer Pair 2
Forward primer: AGTGGAGAGGGTGAAGGTGA
Reverse primer: TACATAACCTTCGGGCATGG
Sequence start and end point: 113 and 304

1
61
121
181
241
301
361
421
481
541
601
661
721
781

tacacacgaa
cccaattctt
gggtgaaggt
actacctgtt
aagataccca
tgtacaggaa
caagtttgaa
agatggaaac
catggcagac
agatggaagc
tgtcctttta
cgaaaagaga
catggatgaa
attactaaaa

taaaagataa
gttgaattag
gatgcaacat
ccatggccaa
gatcatatga
agaactatat
ggtgataccc
attcttggac
aaacaaaaga
gttcaactag
ccagacaacc
gaccacatgg
ctatacaaat
tctcagggtt

caaagatgag
atggtgatgt
acggaaaact
cacttgtcac
aacagcatga
ttttcaaaga
ttgttaatag
acaaattgga
atggaatcaa
cagaccatta
attacctgtc
tccttcttga
aaatgtccag
cctggttaaa

841 agattcatta aaattgtatg aataatttat tgatgttatt gatagaggtt attttcttat

901 taaacaggct acttggagtg tattcttaat tctatattaa ttacaatttg atttgacttg
961 ctcaaa

Primer Pair 3
Forward primer: TCAGTGGAGAGGGTGAAGGT
Reverse primer: TACATAACCTTCGGGCATGG
Sequence start and end point: 111 and 304

1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961

tacacacgaa
cccaattctt
gggtgaaggt
actacctgtt
aagataccca
tgtacaggaa
caagtttgaa
agatggaaac
catggcagac
agatggaagc
tgtcctttta
cgaaaagaga
catggatgaa
attactaaaa
agattcatta
taaacaggct
ctcaaa

taaaagataa
gttgaattag
gatgcaacat
ccatggccaa
gatcatatga
agaactatat
ggtgataccc
attcttggac
aaacaaaaga
gttcaactag
ccagacaacc
gaccacatgg
ctatacaaat
tctcagggtt
aaattgtatg
acttggagtg

caaagatgag
atggtgatgt
acggaaaact
cacttgtcac
aacagcatga
ttttcaaaga
ttgttaatag
acaaattgga
atggaatcaa
cagaccatta
attacctgtc
tccttcttga
aaatgtccag
cctggttaaa
aataatttat
tattcttaat

taaaggagaa
taatgggcac
tacccttaaa
tactttctct
ctttttcaag
tgacgggaac
aatcgagtta
atacaactat
agttaacttc
tcaacaaaat
cacacaatct
gtttgtaaca
acttccaatt
ttcaggctga
tgatgttatt
tctatattaa

gaacttttca
aaattttctg
tttatttgca
tatggtgttc
agtgccatgc
tacaagacac
aaaggtattg
aactcacaca
aaaattagac
actccaattg
gccctttcga
gctgctggga
gacactaaag
gatattattt
gatagaggtt
ttacaatttg

ctggagttgt
tcagtggaga
ctactggaaa
aatgcttttc
ccgaaggtta
gtgctgaagt
attttaaaga
atgtatacat
acaacattga
gcgatggccc
aagatcccaa
ttacacatgg
tgtccgaaca
atatatttat
attttcttat
atttgacttg

taaaggagaa
taatgggcac
tacccttaaa
tactttctct
ctttttcaag
tgacgggaac
aatcgagtta
atacaactat
agttaacttc
tcaacaaaat
cacacaatct
gtttgtaaca
acttccaatt
ttcaggctga

gaacttttca
aaattttctg
tttatttgca
tatggtgttc
agtgccatgc
tacaagacac
aaaggtattg
aactcacaca
aaaattagac
actccaattg
gccctttcga
gctgctggga
gacactaaag
gatattattt

ctggagttgt
tcagtggaga
ctactggaaa
aatgcttttc
ccgaaggtta
gtgctgaagt
attttaaaga
atgtatacat
acaacattga
gcgatggccc
aagatcccaa
ttacacatgg
tgtccgaaca
atatatttat

Primer Pair 4
Forward primer: GTCCACACAATCTGCCCTTT
Reverse primer: GCCTGAATTTAACCAGGAACC
Sequence start and end point: 628 and 817

1
61
121
181
241
301
361
421
481
541
601
661
721
781

tacacacgaa
cccaattctt
gggtgaaggt
actacctgtt
aagataccca
tgtacaggaa
caagtttgaa
agatggaaac
catggcagac
agatggaagc
tgtcctttta
cgaaaagaga
catggatgaa
attactaaaa

taaaagataa
gttgaattag
gatgcaacat
ccatggccaa
gatcatatga
agaactatat
ggtgataccc
attcttggac
aaacaaaaga
gttcaactag
ccagacaacc
gaccacatgg
ctatacaaat
tctcagggtt

caaagatgag
atggtgatgt
acggaaaact
cacttgtcac
aacagcatga
ttttcaaaga
ttgttaatag
acaaattgga
atggaatcaa
cagaccatta
attacctgtc
tccttcttga
aaatgtccag
cctggttaaa

841 agattcatta aaattgtatg aataatttat tgatgttatt gatagaggtt attttcttat

901 taaacaggct acttggagtg tattcttaat tctatattaa ttacaatttg atttgacttg
961 ctcaaa

Primer Pair 5
Forward primer: GGTGATGCAACATACGGAAA
Reverse primer: TACATAACCTTCGGGCATGG
Sequence start and end point: 128 and 304

1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961

tacacacgaa
cccaattctt
gggtgaaggt
actacctgtt
aagataccca
tgtacaggaa
caagtttgaa
agatggaaac
catggcagac
agatggaagc
tgtcctttta
cgaaaagaga
catggatgaa
attactaaaa
agattcatta
taaacaggct
ctcaaa

taaaagataa
gttgaattag
gatgcaacat
ccatggccaa
gatcatatga
agaactatat
ggtgataccc
attcttggac
aaacaaaaga
gttcaactag
ccagacaacc
gaccacatgg
ctatacaaat
tctcagggtt
aaattgtatg
acttggagtg

caaagatgag
atggtgatgt
acggaaaact
cacttgtcac
aacagcatga
ttttcaaaga
ttgttaatag
acaaattgga
atggaatcaa
cagaccatta
attacctgtc
tccttcttga
aaatgtccag
cctggttaaa
aataatttat
tattcttaat

taaaggagaa
taatgggcac
tacccttaaa
tactttctct
ctttttcaag
tgacgggaac
aatcgagtta
atacaactat
agttaacttc
tcaacaaaat
cacacaatct
gtttgtaaca
acttccaatt
ttcaggctga
tgatgttatt
tctatattaa

gaacttttca
aaattttctg
tttatttgca
tatggtgttc
agtgccatgc
tacaagacac
aaaggtattg
aactcacaca
aaaattagac
actccaattg
gccctttcga
gctgctggga
gacactaaag
gatattattt
gatagaggtt
ttacaatttg

ctggagttgt
tcagtggaga
ctactggaaa
aatgcttttc
ccgaaggtta
gtgctgaagt
attttaaaga
atgtatacat
acaacattga
gcgatggccc
aagatcccaa
ttacacatgg
tgtccgaaca
atatatttat
attttcttat
atttgacttg

Bibliography
(n.d.). Retrieved from http://bioinfo.ut.ee/cgi-bin/primer3-0.4.0/primer3_results.cgi
(n.d.). Retrieved from National Center of Biological Information: ncbi.nlm.nih.gov
Aequorea victoria green-fluorescent protein mRNA, complete cds. (n.d.). Retrieved
from National Center for Biological Information -NCBI:
http://www.ncbi.nlm.nih.gov/nuccore/M62653