Goal: Construct PHIX179 promoter linkers with SphI and NheI cloning sites to the fore and aft.  Digestion of pBEST-OR1OR1Pr-UTR1-deGFP-T500 will remove OR1OR2Pr, at which point SphI-PX-NheI will be ligated into the OR1OR2Pr position, creating pBEST-PX-UTR1-deGFP-T500. Extract promoter (and reverse compliment) from PHIX179 sequence. Attach above cloning sites, and then remove appropriate number of bases on each end to create correct post-hybridization sticky ends. PB-UTRB and PG-UTRG are too long to synthesize, PCR with overhanging sequences is used instead (see below).

Promoters

PL(*): ATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGT - 61b
PL(*)-PA: ATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGTATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTAATGTCGTCACTGATGCTGCTTCTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAACAATTTCTGGAAAGACGGTAAAGCTGATGGTATTGGCTCTAATTTGTCTAGGAAATAACCGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCC - 281 b
PA: GTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCC - 42b (includes 7 bases unmapped intergenic sequence upstream from accepted PA)
PB: TTGCAAAATACGTGGCCTTATGGTTACAGTATGCCC - 36 b
PD: GTTGACATTTTAAAAGAGCGTGGATTACTATCTGAGTCC - 39 b
PF: TTGGTATGTAGGTGGTCAACAATTTTAATTGCAGGG - 36 b
PG: GTGATAAAAGATTGAGTGTGAGGTTATAACGCCGAAG - 37 b

SphI: GCATG^C
NheI: G^CTAGC  

Constructions ...

1. Promoterless negative control: SphI-HindIII-NheI (18b) (?)
GCATGCGGATCCGCTAGC 
Sense linker (8b)
CGGATCCG
Antisense linker (16b)
CTAGCGGATCCGCATG

2. OR1OR2Pr Positive control: pBEST-OR1OR2Pr-UTR1-deGFP-T500 (already constructed)

3. SphI-PL-NheI (73b)
GCATGCATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGTGCTAGC
Sense linker (63b)
CATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGTG
Antisense linker (71b)
CTAGCACCGCTGATTCTGCGTTTGCTGATGAACTAAGTCAACCTCAGCACTAACCTTGCGAGTCATGCATG

4. SphI-PA-NheI (55b)
GCATGCCGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCGCTAGC
Sense linker (45b)
CCGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCG
Antisense linker (53b)
CTAGCGGAGGCATGAAAACATACAATTGGGAGGGTGTCAATCCTGACGGCATG

5. SphI-PB-NheI (48b)
GCATGCTTGCAAAATACGTGGCCTTATGGTTACAGTATGCCCGCTAGC
Sense linker (38b)
CTTGCAAAATACGTGGCCTTATGGTTACAGTATGCCCG
Antisense linker (46b)
CTAGCGGGCATACTGTAACCATAAGGCCACGTATTTTGCAAGCATG

6. SphI-PD-NheI (51b)
GCATGCGTTGACATTTTAAAAGAGCGTGGATTACTATCTGAGTCCGCTAGC
Sense linker (41b)
CGTTGACATTTTAAAAGAGCGTGGATTACTATCTGAGTCCG
Antisense linker (49b)
CTAGCGGACTCAGATAGTAATCCACGCTCTTTTAAAATGTCAACGCATG

7. SphI-PF-NheI (48b)
GCATGCTTGGTATGTAGGTGGTCAACAATTTTAATTGCAGGGGCTAGC
Sense linker (38b)
CTTGGTATGTAGGTGGTCAACAATTTTAATTGCAGGGG
Antisense linker (46b)
CTAGCCCCTGCAATTAAAATTGTTGACCACCTACATACCAAGCATG

8. SphI-PG-NheI (49b)
GCATGCGTGATAAAAGATTGAGTGTGAGGTTATAACGCCGAAGGCTAGC
Sense linker (39b)
CGTGATAAAAGATTGAGTGTGAGGTTATAACGCCGAAGG
Antisense linker (47b)
CTAGCCTTCGGCGTTATAACCTCACACTCAATCTTTTATCACGCATG

9. SalI-SphI-PL-PA-NheI-EcoR1 (281b)
ATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGGTATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTAATGTCGTCACTGATGCTGCTTCTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAACAATTTCTGGAAAGACGGTAAAGCTGATGGTATTGGCTCTAATTTGTCTAGGAAATAACCGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCC

Left overhang (SalI-SphI): GTCGACGCATGC
Right overhang (RC NheI-SphI): GCTAGCGAATTC

PRIMER PICKING RESULTS FOR SalI-SphI-PL-PA-NheI-EcoR1

OLIGO            start  len      tm     gc%   any    3' seq 
LEFT PRIMER          1   19   53.74   47.37  4.00  0.00 ATGACTCGCAAGGTTAGTG
RIGHT PRIMER       281   19   54.38   42.11  4.00  0.00 GGAGGCATGAAAACATACA
SEQUENCE SIZE: 281
INCLUDED REGION SIZE: 281

PRODUCT SIZE: 281, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00

    1 ATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACGCAGAATCAGCGG
      >>>>>>>>>>>>>>>>>>>                                         

   61 TATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTAATGTCGTCACTGATGCT
                                                                  

  121 GCTTCTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAAC
                                                                  

  181 AATTTCTGGAAAGACGGTAAAGCTGATGGTATTGGCTCTAATTTGTCTAGGAAATAACCG
                                                                  

  241 TCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCC
                            <<<<<<<<<<<<<<<<<<<

Left: GTCGACGCATGCATGACTCGCAAGGTTAGTG
Right: GCTAGCGAATTCGGAGGCATGAAAACATACA
----------------

Ordered 5/3/12.