LOCUS       sequence-474608-        6418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpress Sc Rad27 (FEN1) (C-terminal S6 tag) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6418)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 6418)
  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"
COMMENT     Sequence Label: Sc Rad27-S6- pET24
FEATURES             Location/Qualifiers
     source          1..6418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(498..1580)
                     /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"
     primer_bind     1542..1561
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1581..1658)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1864..1883
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1967..1986
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1967..1985
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1986..2010
                     /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             2025..2047
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3207..3224
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3319..3337)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3333..3380
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3417..3872
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3504..3523)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3714..3735
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3964..4779)
                     /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(3964..4779)
                     /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     4687..4706
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4771..4790
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4901..5489
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5390..5409
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5643..5660
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5675..5814
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5740..5759)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5900..5922
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5916..6107)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
       61 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
      121 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
      181 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
      241 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
      301 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
      361 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
      421 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
      481 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
      541 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
      601 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
      661 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
      721 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
      781 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
      841 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
      901 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
      961 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     1021 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     1081 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     1141 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     1201 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     1261 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     1321 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     1381 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     1441 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     1501 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     1561 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     1621 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     1681 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     1741 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     1801 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     1861 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     1921 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     1981 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     2041 aaggagatat acatatgggt attaaaggtt tgaatgcaat tatatcggaa catgttccct
     2101 ctgctatcag gaaaagcgat atcaagagct tttttggcag aaaggttgcc atcgatgcct
     2161 ctatgtctct atatcagttt ttaattgctg taagacagca agacggtggg cagttgacca
     2221 atgaagccgg tgaaacaacg tcacacttga tgggtatgtt ttataggaca ctgagaatga
     2281 ttgataacgg tatcaagcct tgttatgtct tcgacggcaa acctccagat ttgaaatctc
     2341 atgagttgac aaagcggtct tcaagaaggg tggaaacaga aaaaaaactg gcagaggcaa
     2401 caacagaatt ggaaaagatg aagcaagaaa gaagattggt gaaggtctca aaagagcata
     2461 atgaagaagc ccaaaaatta ctaggactaa tgggaatccc atatataata gcgccaacgg
     2521 aagctgaggc tcaatgtgct gagttggcaa agaagggaaa ggtgtatgcc gcagcaagtg
     2581 aagatatgga cacactctgt tatagaacac ccttcttgtt gagacatttg actttttcag
     2641 aggccaagaa ggaaccgatt cacgaaatag atactgaatt agttttgaga ggactcgact
     2701 tgacaataga gcagtttgtt gatctttgca taatgcttgg ttgtgactac tgtgaaagca
     2761 tcagaggtgt tggtccagtg acagccttaa aattgataaa aacgcatgga tccatcgaaa
     2821 aaatcgtgga gtttattgaa tctggggagt caaacaacac taaatggaaa atcccagaag
     2881 actggcctta caaacaagca agaatgctgt ttcttgaccc tgaagttata gatggtaacg
     2941 aaataaactt gaaatggtcg ccaccaaagg agaaggaact tatcgagtat ttatgtgatg
     3001 ataagaaatt cagtgaagaa agagttaaat ctggtatatc aagattgaaa aaaggcttga
     3061 aatctggcat tcagggtagg ttagatgggt tcttccaagt ggtgcctaag acaaaggaac
     3121 agctggctgc tgcggcgaaa agagcacaag aaaataaaaa attgaacaaa aataagaata
     3181 aagtcacaaa gggaagaaga ctcgagcacc accaccacca ccactcttct ggtgattctt
     3241 tgtcttggtt gttgagattg ttgaattgag atccggctgc taacaaagcc cgaaaggaag
     3301 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     3361 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     3421 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3481 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3541 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3601 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3661 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3721 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3781 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3841 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     3901 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     3961 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     4021 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     4081 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     4141 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     4201 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     4261 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     4321 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     4381 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     4441 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     4501 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     4561 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     4621 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     4681 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     4741 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     4801 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4861 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4921 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4981 ttcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5041 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5101 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5161 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5221 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5281 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5341 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5401 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5461 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5521 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5581 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5641 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     5701 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     5761 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     5821 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     5881 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     5941 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     6001 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     6061 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     6121 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     6181 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     6241 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     6301 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     6361 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccga
//
