Knight:pRML1/2-sequencing

Sequencing primers for pRML1, pRML2:

• T3 for pRML1
• T7 for pRML2

For both pRML1 & 2:

• attgtctcatgagcggatac,ampR-F
• ggtcacgctgcgcgtaacc,F1-F (works on pRML1, missing region on pRML2)
• atggaaaaacgccagcaacg,322ori-R

Construction primers:

• pRML1 (Left YAC arm)
  • GTT TCT TCC TGC AGC GGC CGC TAC TAG TA AGCTGGGCCATTCTCATGAAG,pRML1-R
  • biobrick right end, binding site for Cen4 sequence
  • binds with stutter
• pRML2-F (Right YAC arm)
  • GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ACGCGCGTCGACGGTATCGG,pRML2-F
  • biobrick left end, ARSH4 binding site
  • binds with stutter

pRML1

>pRML1-T3 710 bp
GTCGACTCGCGAGCTAGCTTCGAAATCGATGAATTTCCGCGCGAGGCAGACCCCAACCCC
AACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCC
CAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCC
CAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCC
CAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCC
CAACCCCAACCCCAATAATTTCTTTGACATTGAGTAAAAGTTATTTATTGAATGTAATAC
TTTGATTTTTAGTTATTTATGAATGATTAAGATGTTAAAATGTTTAAATTCTATAATATT
TTNNTTTNTTTATATATGAATAAACATAAANTNTTAATTAAATCTCAAAAATGACTAANN
TAGTCAAANATTGNAGTTCTTATCAAANTTATTTTAATTAANAATTTTATTTTTTGTAGT
TTTCATAAATTTTTATAAGTTTTTCATTCAAAAATAAACTTTTNNAAGGGTTTTTTGTTA
TNAAAGATCAATAAATAAAATGTTTTTCAAGAANTTANTGATGATTAATAAATTTTATCT
AAAAATGAAGCTTATCGCCTCGACCTGCANGNATGCCTGCATGTCGATCG

• pYAC4: 50-676 (both directions) CTAG missing, 5 c's
• 1-10 T7 expression vector pNAM
• regions before and at end not found.

---

>pRML1-ampR-F 971 bp
AAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAATTCCCGCCC
GTAGCGCGCGCGCACCAGCGCGGCATCGCACCCGAGCACCAGCTCCCCCGTCGTCCAGAT
GCCCACGGGCCACGTCGAGGCCGACGGGGAGAAATACACGTACCTACCTGGGGATCTCAA
CAGGCCCCGGGTGGCCAACCAGGTCGTGGACGCGTTGTGCAGGTGCGTGATGTCCAGCTC
CGTCGTCGGGTGCCGCCGGGCCCCAACCGGCGGTCGGGGGGGCGGTGTATCACGCGGCCC
GCTCGGGTGGCTCGCCGTCGCCACGTTGTCTCCCCGCGGGAACGTCAGGGCCTCGGGGTC
AGGGACGGCCGAAAACGTTACCCAGGCCCGGGAACGCAGCAACACGGAGGCGGCTGGATT
GTGCAAGAGACCCTTAAGGGGGGCGACCGAGGGGGGAGGCTGGGCGGTCGGCTCGACCGT
GGTGGGGGCGGGCAGGCTCGCGTTCGGGGGCCGGCCGAGCAGGTAGGTCTTCGGGATGTA
AAGCAGCTGGCCGGGGTCCCGCGGAAACTCGGCCGTGGTGACCAATACAAAACAAAAGCG
CTCCTCGTACCAGCGAAGAAGGGGCAGAGATGCCGTAGTCAGGTTTAGTTCGTCCGGCGG
CGCCAGAAATCCGCGCGGTGGTTTTTGGGGGTCGGGGGTGTTTGGCAGCCACAGACGCCC
GGTGTTCGTGTCGCGCCAGTACATGCGGTCCATGCCCAGGCCATCCAAAAACCATGGGTC
TGTCTGCTCAGTCCAGTCGTGGACCTGACCCCACGCAACGCCCAAAATAATAACCCCCAC
GAACCATAAACCATTCCCCATGGGGGACCCCGTNCCTAACCCACGGGGCCCGTGGCTATG
GCAGGGCTTGCCGCCCCGACGTTGGCTGCNAGCCCTGGCCTTCACCCGANTTGGGGTTGG
GGTGGGAAAAG

• pBluescript: 1-47
• Thymidine kinase 50-end

---

>pRML1-322ori-R 712 bp
CTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAA
CCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAG
CGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCT
GTGCGGTATTTCACACCGCATACGGTACCCGGGGATCCATATGACTAGTGATCCTCTAGA
GTCGATCGACCTGCAGGCATGCCTGCAGGTCGAGGCGATAAGCTTCATTTTTAGATAAAA
TTTATTAATCATCATTAATTTCTTGAAAAACATTTTATTTATTGATCTTTTATAACAAAA
AACCCTTCTAAAAGTTTATTTTTGAATGAAAAACTTATAAAAATTTATGAAAACTACAAA
AAATAAAATTTTTAATTAAAATAATTTTGATAAGAACTTCAATCTTTGACTAGCTTAGTC
ATTTTTGAGATTTAATTAATATTTTATGTTTATTCATATATAAACTATTCAAAATATTAT
AGAATTTAAACATTTTAACATCTTAATCATTCATAAATAACTAAAAATCAAAGTATTACA
TTCAATAAATAACTTTTACTCAATGTCAAAGAAATTATTGGGGTTGGGGTTGGGGTTGGG
GTTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG

• 1-152 pluescript
• perfect inverted repeat 246-273 TCGACCTGCAGGCATGCCTGCAGGTCGA
• 1-202 pYAC4
• 277-712 pYAC4 telomere end

---

>pRML1-wh 286 bp
TTTTTTTCTTTTTTATAAGAATGTTACCACCTAAATAAGTTATAAAGTCAATAGTTAAGT
TTGATATTTGATTGTAAAATACCGTAATATATTTGCATGATCAAAAGGCTCAATGTTGAC
TAGCCAGCATGTCAACCACTATATTGATCACCGATATtaGGACTTCCACACCAACTAGct
agTAATATGACAATAAATTCAAGATATTCTTCATGAGAATGGCCCAGCTCATGTTTGACA
GCTTATCATCGCTACGTAAATTAACCCTCACTAAAGGGAATTCAGA

• Not data; whitehead reported sequence
• Cen4 prior to 240
• bacterial begins 230
• T3 primer ATTAACCCTCACTAAAGGGA, overlaps EcoRI site
• construction primer (not working) is CTTCATGAGAATGGCCCAGCT (reverse) 209-229

---

New primers 9/17/06

ACCTGACTACGGCATCTCTG,TKR1 (46160)

CACGAACCATAAACCATTCC,TK1 (46360)

ttgcctgggctttggacgtc,TKR2 (46860)

cagataccgcaccgtattgg,TK2 (47060)

caccacacaacaccgcctcg,TKR3 (47570)

tggtcgaacgcagacgcgtg,TK3 (47760)

cacaggtagttctggtccattg,TRP1 (460) pSH63 AF298789

agcatcggaatctagagcac,TRPR1 (550)

ggtgttgatgtaagcggagg,TRP2 (1060)

ctatttcttagcatttttgacg,TRPR2 (1130)

acatcaacaccaataacgcc,YAC4R1 (9710) pYAC4

gcttacattt tatgttagct gg,YAC4-1 (9640)

agcgttgcct catcaatgcg,YAC4-2 (10370)

cacacagtggaccgaacgtg,YAC4R2

GGAAATTCATCGATTTCGAAG,T3R

September 24 primers:

GCTCAATGTTGACTAGCCAGC,CENF

TGATCGCGTAGTCGATAGTGG,TRPR0

GCGGCACGCTGTTGACGCTG,TK4

pRML2

Sequencing results 9/8/06:

---

>pRML2-322ori-R
CTGGCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGG
ATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC
GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCG
CGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA
GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACT
TTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAA
ACAGCTATGACCATGATTACGCCAAGCGCGGGCCGCTCTAGAACTAGTGGATCCTCTAGA
GTCGATCGACCTGCAGGCATGCCTGCAGGTCGAGGCGATAAGCTTCATTTTTAGATAAAA
TTTATTAATCATCATTAATTTCTTGAAAAACATTTTATTTATTGATCTTTTATAACAAAA
AACCCTTCTAAAAGTTTATTTTTGAATGAAAAACTTATAAAAATTTATGAAAACTACAAA
AAATAAAATTTTTAATTAAAATAATTTTGATAAGAACTTCAATCTTTGACTAGCTTAGTC
ATTTTTGAGATTTAATTAATATTTTATGTTTATTCATATATAAACTATTCAAAATATTAT
AGAATTTAAACATTTTAACATCTTAATCATTCATAAATAACTAAAAATCAAAGTATTACA
TTCAATAAATAACTTTTACTCAATGTCAAAGAAATTATTGGGGTTGGGGTTGGGGTTGGG
GTTGGGGTT

• pBluescript 5-390
• contains M13-rev, lac promoter (omits T3 site)
• 457-end pYAC4 telomere

---

>pRML2-F1-F
TCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTA
CGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTT
TCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGAATTAATTCGATATCAAGCT
TTTCAATTCATCATTTTTTTTTTATTCTTTTTTTTGATTTCGGTTTCCTTGAAATTTTTT
TGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGT
ATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACT
GCACAGAACAAAAACCTGCAGGAAACGAAGATAAATCATGTCGAAAGCTACATATAAGGA
ACGTGCTGCTACTCATCCTAGTCCTGTTGCTGCCAAGCTATTTAATATCATGCACGAAAA
GCAAACAAACTTGTGTGCTTCNTTGGATGTTCGTACCACCAAGGAATTACTGGAGTTAGT
TGAAGCATTAGGTCCCAAAATTTGTTTACNAAAAACACATG

• pBluescript: 1-160
• URA3: 182-end

---

>pRML2-ampR-F
TTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAA
ATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAA
AATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAA
CAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA
GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCG
TAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCC
GGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGC
AAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACA
GGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGC
CTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGT
AACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGAATTAAT
TCGATATCAAGCTTTTCAATTCATCATTTTTTTTTTATTCTTTTTTTTGATTTCGGTTTC
CTTGAAATTTTTTTGATTCGGTAANCTCCGAACAGAAGGAAGAACGAANGAANGGAGCAC
AGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAAGAAACATGAAATTGCCNAGT
ATTCTTAACCCAACTGCACAGAACAAAAACCTGCAGGAAANNNANATAAATCATGTCNAA
AGCTACATATAAGGAACGTGCTGCTACTNCATNCCTAGTCCTGTTGNNTGCCAAGCTATT
TTNATATNATGCCCGAAAAGCAAACAANCTTGTGTGCTTCATTGGATNTNCGNANCCACC
AAGG

• pBluescript SK(+) 1-653
• M13F 615-637
• M13F20: 630-646
• LacZa: 644-489
• F1 ori: 471-165
• end: URA3

---

>pRML2-T7 352 bp
TCAGATCTGCGGCCGCGGTCGACTCGCGAGCTAGCTTCGAAATCGATGAATTTCCGCGCG
AGGCAGACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCA
ACCCCAACCCCAACCCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCC
AACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCC
AACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCC
AACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCTTTAATTTTT

• 69-342: telomere repeat
• 9-35: T7 expression vector part

---

>pRML2-wh 260 bp
AAAAAGCGTACTTTACATATATATTTATTAGACAAGAAAAGCAGATTAAATAGATATACA
TTCGATTAACGATAAGTAAAATGTAAAATCACAGGATTTTCGTGTGTGGTCTTCTACACA
GACAAGATGAAACAATTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGATAAAAGGTAG
TATTTGTTGGCGATCCCCCGATACCGTCGACGCGCGTAATACGACTCACTATAGGGCGAA
TTGGGTACATCGAATTCAGA

• Sequence from Whitehead site, not real data
• ARSH4 sequence prior to 200
• bacterial sequence starts 200
• test primer sequence CCGTCGACGCGCGT (reverse)

---

>merged whitehead/T7 sequence region 607 bp
AAAAAGCGTACTTTACATATATATTTATTAGACAAGAAAAGCAGATTAAATAGATATACA
TTCGATTAACGATAAGTAAAATGTAAAATCACAGGATTTTCGTGTGTGGTCTTCTACACA
GACAAGATGAAACAATTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGATAAAAGGTAG
TATTTGTTGGCGATCCCCCGATACCGTCGACGCGCGTAATACGACTCACTATAGGGCGAA
ATGAATTTCCGCGCGAGGCAGACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACC
CCAACCCCAACCCCAACCCCAACCCCAACCCCCAACCCCAACCCCAACCCCAACCCCAAC
CCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAAC
CCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAAC
CCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCTTT
AATTTTT

• EcoRI dividing line. T7 primer TAATACGACTCACTATAGGG

Primers 9/17/06

aaatt gaagctctaa tttgtgag,URA1 (3200) DQ882849

ggtagagggtgaacgttacag,URA1R (3300)

GGAAATTCATCGATTTCGAAGC,T7R

September 24 primers:

GCAGGCATGCCTGCAGGTCGAG,oriR2

TACCTGAGAGCAGGAAGAGC,ARS