LOCUS       sequence-474602-        6852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpress Sc RFC1 (N-terminal 3X FLAG tag) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6852)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 6852)
  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 FLAG-RFC1-pRSFDuet
FEATURES             Location/Qualifiers
     source          1..6852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     475..494
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     717..734
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        760..851
                     /gene="bla"
                     /label=AmpR promoter
     CDS             852..1667
                     /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             852..1667
                     /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(925..944)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(1897..1944)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1996..2040)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     protein_bind    2149..2173
                     /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)."
     promoter        complement(2174..2192)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(2301..2306,2307..2323,2324..2329)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(2504..2551)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5290..5355)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    5402..5426
                     /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)."
     promoter        complement(5427..5445)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        5491..5568
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5569..6651
                     /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(5588..5607)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      join(6847..6852,1..744)
                     /direction=RIGHT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
ORIGIN
        1 atagctgttc gttgacttga tagaccgatt gattcatcat ctcataaata aagaaaaacc
       61 accgctacca acggtggttt tctcaaggtt cgctgagcta ccaactcttt gaaccaaggt
      121 aagtgggttg gaggaccgca ctcaccaaaa tctgttcttt cagtttagcc ttaacaggtg
      181 cataacttca agacaaagtc ctctaaatca gttaccaatg gctgctgcca gtggcgataa
      241 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      301 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      361 ataccaacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      421 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      481 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tggcttgagc gtcgattttt
      541 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc tgcggcgttg gcttcttccg
      601 gtgctttgct ttttgctcac atgttctttc cggctttatc ccctgattct gtggataacc
      661 gtattaccgc ttttgagtga gctgacaccg ctcgccgcag tcgaacgacc gagcgtagcg
      721 agtcagtgag cgaggaagcg gaagagcgct gcatgcctat ttgtttattt ttctaaatac
      781 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
      841 aaaggaagag tatgagccat attcaacggg aaacgtcttg ctctaggccg cgattaaatt
      901 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
      961 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     1021 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     1081 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     1141 tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag
     1201 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     1261 gtaattgtcc ttttaacagc gaccgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     1321 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     1381 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     1441 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     1501 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     1561 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     1621 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa ttaattcatg
     1681 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     1741 ccccgaaaag tgccacttgc ggagacccgg tcgtcagctt gtcgtcggtt cagggcaggg
     1801 tcgttaaata gccgcttatg tctattgctg gtttaccggt ttattgacta ccggaagcag
     1861 tgtgaccgtg tgcttctcaa atgcctgagg tttcagcaaa aaacccctca agacccgttt
     1921 agaggcccca aggggttatg ctagttattg ctcagcggtg gcagcagcct aggttaatta
     1981 agctgcgcta gtagacgagt ccatgtgctg gcgttcaaat ttcgcagcag cggtttcttt
     2041 accagactcg agggtaccga cgtcagcgat cgcgtggccg gccgatatcc aattgagatc
     2101 tgccatatgt atatctcctt cttatactta actaatatac taagatgggg aattgttatc
     2161 cgctcacaat tcccctatag tgagtcgtat taatttcgat tatgcggccg tgtacaatac
     2221 gattactttc tgttcgactt aagcattatg cggccgcaag cttgtcgacc tgcaggcgcg
     2281 ccgagctcga attctcatgt ttgacagctt atcatcgata agctttaatg cggtagttta
     2341 tcacagttaa attgctaacg cagtcaggca ccgtgtatga aatctaacaa tgcgctcatc
     2401 gtcatcctcg gcaccgtcac cctggatgct gtaggcatag gcttggttat gccggtactg
     2461 ccgggcctct tgcgggatat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
     2521 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact
     2581 cagcttcctt tcgggctttg ttagcagccg gatcaccaca cagttttcgt atctgtgata
     2641 gaacggctca caaaatcaat gagaagaaaa gtgtaattat aatcttagtg tatgaataaa
     2701 tcatgctttc gttttccttt ttttgctacc accaggttta ctggtggcag tcttcctctt
     2761 cgtaggtttg gctttctgct tgataagttt atccttcttc aagtctgtgc tactatcttg
     2821 tgtttcttca tcatctgcgg gaactggatt gtcatctgcg tcaacgacat cttcgaaatc
     2881 gggggtgcta gtactggtgc caacaccacc accaccaata gtactacctg ttctgtaaat
     2941 tgcgactgga tgtgtcatac tgttgtattt ccgcgtgaat ccacttttaa ccgtagctgg
     3001 tatctttttg ataatggctg tggtcacatc aggacctacg aaaaactcca taatagaatc
     3061 ccagtcttct ttggtcaaat agtaatcgtc cattacctct atgacagatg aaattgcatc
     3121 agcaccttgc ttcaaaaatg ggtccaataa tctttttcta aaagttggta ggtaatccag
     3181 ccttagtcca attttatcag tagatgtacc tagtcttgta tgataatgta tctcttgcag
     3241 taacctataa tatttggcag atttagaatt ttggcccaac caagctgtga aattgattct
     3301 tcccgccata tggccggcaa cttttgatgc tggataaaca gatgaaagaa cagcatgcaa
     3361 aggcaaaaga ctccataatt gctcactact acgaatcttc ttttcgacaa tatcacctaa
     3421 tgaaatacag ttagccgcct cagcaactgc ttccaaatgt gactggccgg gtttcaaaac
     3481 gcttggtctt gtagataaat agttttcctg aatcattagg ggtgtgaaat caaagtcatc
     3541 aaaatataat gcgatcttat cattcagtgt aaagttcctt gaacctatgt ctgagtatat
     3601 ttgtccatcc aacattttat gggcaatgtc aaagggtttt agggcaatat tcttttccca
     3661 tgcctttgag atctcgttga tattttcatg gtttatggtt ttagtagtcg tagatatcgt
     3721 tgaaagtaga ttaataacct ggcggatatc acctcttgta gtctgtatca acctatcaat
     3781 gacatttgga tcaagtttga acttttctct gatcgcaatt gtcattaatc ttgatttgat
     3841 actgttagca tcaggtcttc taaattggat atcaagacat actctatcga atggtctcat
     3901 ttttggtaaa ttacgttcat tacaaattag aatcaatgga gttgacgtct tacgacaaaa
     3961 ctgtgccagc tggccaactc cccccctatc gcctccgctc ataccatcaa cttcatccat
     4021 gataatgaca aaatgtttcc cgttcaaatt ttgagcttct tcgttgtgtt tgaaatatcc
     4081 cactacagac atgttatcaa gagcgttttt aacaccggca ttaagtaaag ttttagagcg
     4141 aacatcagaa gcgttttgtt caagaatgtc atacccaagc tcttgtgcaa ctaaatgagc
     4201 agcagttgtc ttcccaatac caggaggacc gtataacatt gcagccctga aaacaccaga
     4261 gccatcttta ccagcatgtt taaaactatt tttctttgaa ttttcccaat tagccaacca
     4321 gttttttaat ttcattacac ttcctttgtt accacagacc tgttgtagat tcgttggtgc
     4381 gtactttact gtccacaatt tatcttcttc cctaaccaca ttgtctctct ccagatggcc
     4441 accagaaact ctagtggctg caagcttctt cgacctttcc tcttctttct taacaagcaa
     4501 ttctgcttct ttggtagcaa tattatgctg ttcttctaat tttcgacgtg ctttttcggc
     4561 agcttccccg tcaccacctt cagcaggcat cccagcaatt aattgcttaa agccttcttc
     4621 gtctatggct ttgattttca attgcttgat tttttctagt ttctttggcc ctgcttcatc
     4681 acctaagact actacagaag ttttgcttga aattgattta gtaacccttg ctccgtatct
     4741 tttcgctaaa gcttccgatg caccacgctc caaggttggc agaacacctg tgaagacaat
     4801 tgttagacct aataaacaat ttggctttcc ttctggaaaa ctacctattt cagaaacaat
     4861 ttcatctgga tccgcatttg aatttgcgct tttgaaatca aatttagcat tctctttcac
     4921 atgaacattt gacagatcca aggatggaat cttatctagt acatcttgag ccgtaatatt
     4981 cgaagccgag tctgaacttt tagaggaggg tttggttttt tttgaacttg ctggctttac
     5041 agttggtgaa gaggccttcc tcttagcggg aagaggtagc tttttttctc cttcaggtgt
     5101 ctctgataca tcaattacat tacttacagg catcttcttt ggtgttttat tggttgattc
     5161 ttggtcagat tcagtatcta agtcgataac ttctggttta gacgaaccca cctgtctcgt
     5221 aggtctggat gttgacgatc ttacggattt cttattttta ccaaagaaat cagaaatatt
     5281 gaccatgtgc ttgtcatcgt catccttgta gtcgatgtca tgatctttat aatcaccgtc
     5341 atggtctttg tagtccatgg tatatctcct tattaaagtt aaacaaaatt atttctacag
     5401 gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc ctaatgcagg
     5461 agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc
     5521 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     5581 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     5641 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     5701 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     5761 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     5821 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     5881 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     5941 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     6001 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     6061 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     6121 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     6181 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     6241 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     6301 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     6361 ggtgcggaca tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     6421 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     6481 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     6541 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     6601 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     6661 aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc
     6721 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actaacatga gaattacaac
     6781 ttatatcgta tggggctgac ttcaggtgct acatttgaag agataaattg cactgaaatc
     6841 tagagtaacg ga
//
