LOCUS       sequence-474607-        6376 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpress Sc Rad27 (FEN1) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6376)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 6376)
  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-pET24
FEATURES             Location/Qualifiers
     source          1..6376
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(138..593)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(275..296)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     487..506
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(630..677)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     673..691
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(744..761)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1921..1943
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1958..1982
                     /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)."
     primer_bind     complement(1982..2001)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1983..2001)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2085..2104)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2310..2387
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2388..3470
                     /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     complement(2407..2426)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4279..4470
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4464..4486)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4572..4711
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4627..4646
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4726..4743)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4897..5485)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4977..4996)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5596..5615)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(5607..6376,1..46)
                     /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             join(5607..6376,1..46)
                     /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     complement(5680..5699)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat taattcatga
       61 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
      121 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
      181 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
      241 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
      301 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
      361 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
      421 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
      481 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
      541 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
      601 gccaatccgg atatagttcc tcctttcagc aaaaaacccc tcaagacccg tttagaggcc
      661 ccaaggggtt atgctagtta ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg
      721 gctttgttag cagccggatc tcagtggtgg tggtggtggt gctcgagtct tcttcccttt
      781 gtgactttat tcttattttt gttcaatttt ttattttctt gtgctctttt cgccgcagca
      841 gccagctgtt cctttgtctt aggcaccact tggaagaacc catctaacct accctgaatg
      901 ccagatttca agcctttttt caatcttgat ataccagatt taactctttc ttcactgaat
      961 ttcttatcat cacataaata ctcgataagt tccttctcct ttggtggcga ccatttcaag
     1021 tttatttcgt taccatctat aacttcaggg tcaagaaaca gcattcttgc ttgtttgtaa
     1081 ggccagtctt ctgggatttt ccatttagtg ttgtttgact ccccagattc aataaactcc
     1141 acgatttttt cgatggatcc atgcgttttt atcaatttta aggctgtcac tggaccaaca
     1201 cctctgatgc tttcacagta gtcacaacca agcattatgc aaagatcaac aaactgctct
     1261 attgtcaagt cgagtcctct caaaactaat tcagtatcta tttcgtgaat cggttccttc
     1321 ttggcctctg aaaaagtcaa atgtctcaac aagaagggtg ttctataaca gagtgtgtcc
     1381 atatcttcac ttgctgcggc atacaccttt cccttctttg ccaactcagc acattgagcc
     1441 tcagcttccg ttggcgctat tatatatggg attcccatta gtcctagtaa tttttgggct
     1501 tcttcattat gctcttttga gaccttcacc aatcttcttt cttgcttcat cttttccaat
     1561 tctgttgttg cctctgccag ttttttttct gtttccaccc ttcttgaaga ccgctttgtc
     1621 aactcatgag atttcaaatc tggaggtttg ccgtcgaaga cataacaagg cttgataccg
     1681 ttatcaatca ttctcagtgt cctataaaac atacccatca agtgtgacgt tgtttcaccg
     1741 gcttcattgg tcaactgccc accgtcttgc tgtcttacag caattaaaaa ctgatataga
     1801 gacatagagg catcgatggc aacctttctg ccaaaaaagc tcttgatatc gcttttcctg
     1861 atagcagagg gaacatgttc cgatataatt gcattcaaac ctttaatacc catatgtata
     1921 tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt
     1981 cccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct acgccggacg
     2041 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
     2101 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
     2161 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt
     2221 ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga
     2281 gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg
     2341 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     2401 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     2461 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     2521 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     2581 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     2641 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     2701 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     2761 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     2821 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     2881 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     2941 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     3001 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     3061 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     3121 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     3181 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     3241 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     3301 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     3361 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     3421 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     3481 attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc
     3541 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     3601 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc
     3661 ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga
     3721 atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag
     3781 aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg
     3841 ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata
     3901 cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga
     3961 atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc
     4021 attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct
     4081 gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat
     4141 accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc
     4201 cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc
     4261 ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt
     4321 atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag
     4381 gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg
     4441 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     4501 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     4561 gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta
     4621 actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca
     4681 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     4741 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4801 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4861 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4921 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4981 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5041 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5101 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5161 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5221 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5281 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     5341 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5401 ttgaaccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5461 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5521 tcagtggaac gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt
     5581 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta
     5641 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
     5701 atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt
     5761 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac
     5821 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg
     5881 atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag
     5941 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
     6001 attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg
     6061 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg
     6121 gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt
     6181 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa
     6241 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
     6301 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg
     6361 gtattgataa tcctga
//
