LOCUS       sequence-236326-        7773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of the nuclear form of Pif1p (Pif1p-delta40), Nterminal 
            His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7773)
  AUTHORS   Boule JB, Vega LR, Zakian VA
  TITLE     The yeast Pif1p helicase removes telomerase from telomeric DNA.
  JOURNAL   Nature. 2005 Nov 3;438(7064):57-61. Epub 2005 Aug 24.
  PUBMED    16121131
REFERENCE   2  (bases 1 to 7773)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature."; 
            date: "2005-11-3"; volume: "438(7064)"; pages: "57-61. Epub 2005 Aug
            24"
COMMENT     Sequence Label: pET28b-His tagged nuclear Pif1
FEATURES             Location/Qualifiers
     source          1..7773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     563..582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(602..679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     885..904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     988..1007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        988..1006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1007..1031
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1046..1068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1087..1104
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1114..1131
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3625..3642
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3695..3713)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3709..3756
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3793..4248
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3880..3899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4090..4111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4340..5155)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4340..5155)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5063..5082
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5147..5166
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5277..5865
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5766..5785
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6019..6036
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6051..6190
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6116..6135)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6276..6298
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6292..6483)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7292..7773,1..601))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
       61 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      721 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
      781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
      841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
      901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
      961 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1021 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc
     1081 agcagccatc atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatgagt
     1141 agtcgtggtt tcaggtctaa taactttatt caagcacaat tgaagcatcc ttccatactt
     1201 tcaaaagaag acctagattt gctctctgat tcggatgatt gggaagaacc tgattgcata
     1261 cagttagaaa ctgagaagca agaaaagaaa attatcactg acatacataa agaagacccg
     1321 gtggacaaaa agcctatgag ggataaaaat gtcatgaatt ttatcaataa agacagtcct
     1381 ttatcctgga acgatatgtt taaacccagt ataatacaac caccgcagtt aatttctgaa
     1441 aactcatttg accagagcag tcaaaaaaaa tcgagatcga caggattcaa gaatccatta
     1501 agaccagcgt tgaaaaagga aagttctttt gatgaacttc aaaataattc tatatctcaa
     1561 gagagaagtt tggaaatgat aaatgaaaac gaaaagaaga aaatgcaatt tggagaaaag
     1621 attgctgttt tgacgcaaag acctagcttc actgaattgc agaatgacca agatgacagt
     1681 aacttgaatc cccataatgg tgtgaaagtc aagataccga tttgcttaag caaagaacaa
     1741 gaaagtatca tcaagttggc ggaaaatggc cacaacattt tttatacagg gagtgccggt
     1801 accggtaaat ccattctttt acgtgaaatg ataaaagttt taaaaggcat atatggtagg
     1861 gagaatgttg cagtcactgc ttccacgggt ttagctgctt gtaatatcgg tggtataacc
     1921 atacactcgt tcgctggtat aggattagga aaaggtgatg cggataaact ctataaaaaa
     1981 gttcgtaggt ctcgaaagca cctaaggcgc tgggaaaata ttggtgcttt ggttgtcgat
     2041 gaaatatcaa tgttagacgc agaactgctt gataaactcg atttcatagc tagaaaaata
     2101 cggaaaaatc atcaaccctt cggtggaatt caactcatct tctgtggcga ttttttccag
     2161 ttaccgccag tatcaaaaga tcctaataga ccaactaagt ttgctttcga atccaaggct
     2221 tggaaagaag gtgtaaagat gacgattatg ctacaaaagg tttttagaca gcgaggcgat
     2281 gttaagttca ttgacatgtt gaatcggatg agactaggca atattgatga tgaaacagaa
     2341 agagagttca agaagctttc tagaccattg ccagacgatg aaattattcc cgcggaactt
     2401 tatagtacca gaatggaagt agaaagggcc aataattcaa ggctaagtaa attgccaggc
     2461 caggtgcata tttttaatgc aatcgatggc ggtgctttgg aagacgaaga gttaaaggaa
     2521 aggctgttac aaaatttttt agctccaaag gaattacatt tgaaagttgg cgctcaggtt
     2581 atgatggtaa aaaatctaga cgcaacatta gttaatggat cccttggtaa agtcatcgaa
     2641 ttcatggatc cagaaacata tttttgctat gaggcgctaa caaacgatcc atctatgcct
     2701 ccagaaaaac tcgagacttg ggcagaaaac ccttcaaaac taaaagctgc aatggagagg
     2761 gagcaaagtg atggggaaga aagtgcggta gctagtcgca aatcttcagt gaaggaggga
     2821 tttgctaaga gtgatatagg tgagccggtc tctcccctag attcctcagt ttttgacttc
     2881 atgaagagag tcaagacaga tgacgaagtt gtgctggaaa atataaaacg caaggaacaa
     2941 ctgatgcaga ccatacatca aaactctgca ggaaaacgaa ggttacctct cgtgagattc
     3001 aaagcttctg atatgagtac gaggatggtg cttgtcgagc cggaggattg ggcgatagaa
     3061 gacgaaaatg aaaagccact ggtatcaagg gttcaattac cgctaatgct tgcctggtca
     3121 ctatccattc acaaatctca gggtcagaca cttccaaaag ttaaagtgga tttacgtaga
     3181 gtattcgaaa agggtcaggc gtacgttgcc ctttctagag ctgtttcaag agaaggacta
     3241 caggtgttaa attttgacag aactaggatc aaagcacatc aaaaggtaat tgatttttat
     3301 cttactttat cttcagccga aagtgcctat aagcaacttg aggcagatga gcaagtgaaa
     3361 aaaaggaagt tagactacgc accaggccct aaatataagg ctaaatccaa gtcaaagtca
     3421 aattctccag cacccatatc agcgaccaca caatctaata atggtatcgc agcgatgttg
     3481 caaagacaca gtaggaagag atttcagttg aaaaaagagt ctaatagtaa tcaagttcat
     3541 tcattggttt ccgacgaacc tcgtggtcag gataccgaag accacatctt agaataagtc
     3601 gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
     3661 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     3721 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     3781 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3841 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3901 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3961 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4021 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4081 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4141 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4201 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     4261 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4321 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     4381 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     4441 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     4501 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     4561 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     4621 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     4681 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     4741 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     4801 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     4861 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     4921 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     4981 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     5041 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     5101 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     5161 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     5221 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5281 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5341 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5401 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5461 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5521 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5581 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5641 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5701 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5761 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5821 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5881 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5941 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6001 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     6061 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     6121 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     6181 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     6241 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     6301 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     6361 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     6421 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     6481 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     6541 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     6601 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     6661 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     6721 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     6781 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     6841 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     6901 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     6961 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     7021 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     7081 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     7141 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     7201 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     7261 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     7321 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     7381 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     7441 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     7501 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     7561 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     7621 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     7681 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     7741 gctgaatttg attgcgagtg agatatttat gcc
//
