Bioinformatic Analysis Results (NCBI)

• Organism: Stylophora pistillata
• Gene Name: Green Fluorescent Protein (GFP)

• Gene ID: [LOC111319708 GFP-like fluorescent chromoprotein amFP486

• GENE Accession: NW_019220287.1
• mRNA Accession: XM_022922450.1
• Protein Accession: XP_022778185.1

6. Primer Design Results (Primer-BLAST)

The following optimal primer pair was selected for amplifying the GFP gene fragment:

• Product Size: 156 bp.

**RAW OUTPUT PRIMER PICKING RESULTS FOR

Template masking not selected No mispriming library specified Using 1-based sequence positions OLIGO start len tm gc% any_th 3’_th hairpin seq LEFT PRIMER 794 20 58.58 50.00 0.00 0.00 0.00 CCGCAGTTGTTCGTCATGTA RIGHT PRIMER 949 20 58.85 50.00 0.00 0.00 0.00 TCCCAGCCAAGAGTCCTTTT SEQUENCE SIZE: 1063 INCLUDED REGION SIZE: 1063

PRODUCT SIZE: 156, PAIR ANY_TH COMPL: 0.00, PAIR 3’_TH COMPL: 0.00

1 TGGCACCGACCTTGAAGTATGGATTCAAGTGTTTTATGAAGTACCCTGCTGATATACCTG

61 ACTATTTCAAGCTGGCATTTCCCGAAGGTCTAAAATACGACAGAAAAATAACATTTGAAG

121 ATGGAGGGTGTGCCACGGCCACTGTGGAAATGAGGTGAGCATTTCCTTATCAATTAAGTG

181 TTATCAGCTTTCTAACTGGCAGACTGCACAGAGGTCTTTGATAAAAAAGGGAACGTTTCT

241 CATCTGCTTGTGTTGATTTCGCTGTCGGGCTCTACGCATATTATTTTTATTAATGATGAA

301 GGAACGAAAGCAACGGAGAAAGCTCCTTCTTCAAATAATCATTAACTGTGCTACAATTAA

361 TATCAATAATTTATAAAGCGCCAAAAAACTCTTAAGATATCTTCGATTTTATCGAGTCTT

421 TGATATTCTTACCAGTCACTTCAACTTTCTCACATATTTTGCATCATTGCACAAAGATTT

481 CATGGCGACGCGAGAAAGTGAACATTTATGACTGATTTTCATGTTTTTAAGTTACATCAC
541 CGAAGATTAAACCAAAACTGCAGTAAATTGCGTAAATAGGTAAATTATTTTGTTTCCTTA
601 AAGGAATGAGGCAAACCTTAGTTTTCAAAAGTAAACCTCAATTTTTCAGCTTGAAGATTT

661 GCCATTCCGTTTTTATTGGCTTATCCTTGACTATGTTCGTTTCTTTCAGCCCTACTCTTG

721 ATTACTCTTTTTGCTTCAGTCTCATTCCTATTTCAATCTTTCTTCCACGTTAAAAGAACT

781 AAAGATTTTTCTGCCGCAGTTGTTCGTCATGTATTACCCATTCTGTGTTCTGAACTACTT  »»»»»»»»»»
841 TCTACTTGGACAGCCTCAAAGGCAACACCCTTATGCACAAGACCAATTTTCAGGGAGGTA
901 ACTTTCCCATTGACGGCCCTGTCATGCAGAAAAGGACTCTTGGCTGGGAACCAACCTCAG «««««««««« 
961 AGAAAATGACTCCTTGTGATGGAATAATCAAGGGAGACACTATGATGTACCTGATGGTTG

1021 AAGGAGGCAAAACTCTGAAATGCCGATATGAAAACAATTACAG

KEYS (in order of precedence):

left primer ««« right primer

ADDITIONAL OLIGOS start len tm gc% any_th 3’_th hairpin seq

1 LEFT PRIMER 68 20 59.83 55.00 0.00 0.00 0.00 CAAGCTGGCATTTCCCGAAG RIGHT PRIMER 316 20 59.06 50.00 0.00 0.00 0.00 CCGTTGCTTTCGTTCCTTCA PRODUCT SIZE: 249, PAIR ANY_TH COMPL: 0.00, PAIR 3’_TH COMPL: 0.00

2 LEFT PRIMER 1 20 59.02 50.00 0.00 0.00 0.00 TGGCACCGACCTTGAAGTAT RIGHT PRIMER 247 20 57.90 50.00 0.00 1.52 0.00 GCAGATGAGAAACGTTCCCT PRODUCT SIZE: 247, PAIR ANY_TH COMPL: 0.00, PAIR 3’_TH COMPL: 0.00

3 LEFT PRIMER 114 20 58.09 50.00 0.00 0.00 0.00 TTTGAAGATGGAGGGTGTGC RIGHT PRIMER 320 20 58.78 50.00 0.00 0.00 0.00 TTCTCCGTTGCTTTCGTTCC PRODUCT SIZE: 207, PAIR ANY_TH COMPL: 0.00, PAIR 3’_TH COMPL: 0.00

4 LEFT PRIMER 486 20 58.94 50.00 15.93 0.00 0.00 CGACGCGAGAAAGTGAACAT RIGHT PRIMER 669 21 58.47 47.62 0.00 0.00 0.00 CGGAATGGCAAATCTTCAAGC PRODUCT SIZE: 184, PAIR ANY_TH COMPL: 0.00, PAIR 3’_TH COMPL: 0.00

Statistics con too in in not no tm tm high high high high
sid many tar excl ok bad GC too too any_th 3’_th hair- poly end
ered Ns get reg reg GC% clamp low high compl compl pin X stab ok Left 5375 0 0 0 0 1328 0 2589 402 0 0 42 115 0 899 Right 5378 0 0 0 0 1307 0 2641 385 0 0 21 112 0 912 Pair Stats: considered 1821, unacceptable product size 1812, primer in pair overlaps a primer in a better pair 315, ok 9 libprimer3 release 2.4.0

(primer3_results.cgi release 4.1.0)