LOCUS       sequence-481685-        6891 bp    DNA     circular SYN 01-AUG-2025
DEFINITION  Overexpress Sc FLAG-S6-RFC1 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6891)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 6891)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Sc FLAG-S6-RFC1-pRSFDuet
FEATURES             Location/Qualifiers
     source          1..6891
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     381..400
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     623..640
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        666..757
                     /gene="bla"
                     /label=AmpR promoter
     CDS             758..1573
                     /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             758..1573
                     /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(831..850)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(1803..1850)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1902..1946)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     protein_bind    2055..2079
                     /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(2080..2098)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(2207..2212,2213..2229,2230..2235)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(2410..2457)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5232..5297)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    5347..5371
                     /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(5372..5390)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        5436..5513
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5514..6596
                     /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(5533..5552)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      join(6792..6891,1..650)
                     /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 gagctaccaa ctctttgaac caaggtaagt gggttggagg accgcactca ccaaaatctg
       61 ttctttcagt ttagccttaa caggtgcata acttcaagac aaagtcctct aaatcagtta
      121 ccaatggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
      181 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
      241 gagcgaacga cctacaccga actgagatac caacagcgtg agctatgaga aagcgccacg
      301 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
      361 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
      421 cacctctggc ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
      481 aacgcctgcg gcgttggctt cttccggtgc tttgcttttt gctcacatgt tctttccggc
      541 tttatcccct gattctgtgg ataaccgtat taccgctttt gagtgagctg acaccgctcg
      601 ccgcagtcga acgaccgagc gtagcgagtc agtgagcgag gaagcggaag agcgctgcat
      661 gcctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
      721 ctgataaatg cttcaataat attgaaaaag gaagagtatg agccatattc aacgggaaac
      781 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg
      841 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga
      901 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga
      961 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat
     1021 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca
     1081 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct
     1141 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgacc gcgtatttcg
     1201 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga
     1261 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt
     1321 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga
     1381 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga
     1441 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt
     1501 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga
     1561 tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata
     1621 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acttgcggag acccggtcgt
     1681 cagcttgtcg tcggttcagg gcagggtcgt taaatagccg cttatgtcta ttgctggttt
     1741 accggtttat tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc ctgaggtttc
     1801 agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag ttattgctca
     1861 gcggtggcag cagcctaggt taattaagct gcgctagtag acgagtccat gtgctggcgt
     1921 tcaaatttcg cagcagcggt ttctttacca gactcgaggg taccgacgtc agcgatcgcg
     1981 tggccggccg atatccaatt gagatctgcc atatgtatat ctccttctta tacttaacta
     2041 atatactaag atggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat
     2101 ttcgattatg cggccgtgta caatacgatt actttctgtt cgacttaagc attatgcggc
     2161 cgcaagcttg tcgacctgca ggcgcgccga gctcgaattc tcatgtttga cagcttatca
     2221 tcgataagct ttaatgcggt agtttatcac agttaaattg ctaacgcagt caggcaccgt
     2281 gtatgaaatc taacaatgcg ctcatcgtca tcctcggcac cgtcaccctg gatgctgtag
     2341 gcataggctt ggttatgccg gtactgccgg gcctcttgcg ggatatccgg atatagttcc
     2401 tcctttcagc aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt atgctagtta
     2461 ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg gctttgttag cagccggatc
     2521 accacacagt tttcgtatct gtgatagaac ggctcacaaa atcaatgaga agaaaagtgt
     2581 aattataatc ttagtgtatg aataaatcat gctttcgttt tccttttttt gctaccacca
     2641 ggtttactgg tggcagtctt cctcttcgta ggtttggctt tctgcttgat aagtttatcc
     2701 ttcttcaagt ctgtgctact atcttgtgtt tcttcatcat ctgcgggaac tggattgtca
     2761 tctgcgtcaa cgacatcttc gaaatcgggg gtgctagtac tggtgccaac accaccacca
     2821 ccaatagtac tacctgttct gtaaattgcg actggatgtg tcatactgtt gtatttccgc
     2881 gtgaatccac ttttaaccgt agctggtatc tttttgataa tggctgtggt cacatcagga
     2941 cctacgaaaa actccataat agaatcccag tcttctttgg tcaaatagta atcgtccatt
     3001 acctctatga cagatgaaat tgcatcagca ccttgcttca aaaatgggtc caataatctt
     3061 tttctaaaag ttggtaggta atccagcctt agtccaattt tatcagtaga tgtacctagt
     3121 cttgtatgat aatgtatctc ttgcagtaac ctataatatt tggcagattt agaattttgg
     3181 cccaaccaag ctgtgaaatt gattcttccc gccatatggc cggcaacttt tgatgctgga
     3241 taaacagatg aaagaacagc atgcaaaggc aaaagactcc ataattgctc actactacga
     3301 atcttctttt cgacaatatc acctaatgaa atacagttag ccgcctcagc aactgcttcc
     3361 aaatgtgact ggccgggttt caaaacgctt ggtcttgtag ataaatagtt ttcctgaatc
     3421 attaggggtg tgaaatcaaa gtcatcaaaa tataatgcga tcttatcatt cagtgtaaag
     3481 ttccttgaac ctatgtctga gtatatttgt ccatccaaca ttttatgggc aatgtcaaag
     3541 ggttttaggg caatattctt ttcccatgcc tttgagatct cgttgatatt ttcatggttt
     3601 atggttttag tagtcgtaga tatcgttgaa agtagattaa taacctggcg gatatcacct
     3661 cttgtagtct gtatcaacct atcaatgaca tttggatcaa gtttgaactt ttctctgatc
     3721 gcaattgtca ttaatcttga tttgatactg ttagcatcag gtcttctaaa ttggatatca
     3781 agacatactc tatcgaatgg tctcattttt ggtaaattac gttcattaca aattagaatc
     3841 aatggagttg acgtcttacg acaaaactgt gccagctggc caactccccc cctatcgcct
     3901 ccgctcatac catcaacttc atccatgata atgacaaaat gtttcccgtt caaattttga
     3961 gcttcttcgt tgtgtttgaa atatcccact acagacatgt tatcaagagc gtttttaaca
     4021 ccggcattaa gtaaagtttt agagcgaaca tcagaagcgt tttgttcaag aatgtcatac
     4081 ccaagctctt gtgcaactaa atgagcagca gttgtcttcc caataccagg aggaccgtat
     4141 aacattgcag ccctgaaaac accagagcca tctttaccag catgtttaaa actatttttc
     4201 tttgaatttt cccaattagc caaccagttt tttaatttca ttacacttcc tttgttacca
     4261 cagacctgtt gtagattcgt tggtgcgtac tttactgtcc acaatttatc ttcttcccta
     4321 accacattgt ctctctccag atggccacca gaaactctag tggctgcaag cttcttcgac
     4381 ctttcctctt ctttcttaac aagcaattct gcttctttgg tagcaatatt atgctgttct
     4441 tctaattttc gacgtgcttt ttcggcagct tccccgtcac caccttcagc aggcatccca
     4501 gcaattaatt gcttaaagcc ttcttcgtct atggctttga ttttcaattg cttgattttt
     4561 tctagtttct ttggccctgc ttcatcacct aagactacta cagaagtttt gcttgaaatt
     4621 gatttagtaa cccttgctcc gtatcttttc gctaaagctt ccgatgcacc acgctccaag
     4681 gttggcagaa cacctgtgaa gacaattgtt agacctaata aacaatttgg ctttccttct
     4741 ggaaaactac ctatttcaga aacaatttca tctggatccg catttgaatt tgcgcttttg
     4801 aaatcaaatt tagcattctc tttcacatga acatttgaca gatccaagga tggaatctta
     4861 tctagtacat cttgagccgt aatattcgaa gccgagtctg aacttttaga ggagggtttg
     4921 gttttttttg aacttgctgg ctttacagtt ggtgaagagg ccttcctctt agcgggaaga
     4981 ggtagctttt tttctccttc aggtgtctct gatacatcaa ttacattact tacaggcatc
     5041 ttctttggtg ttttattggt tgattcttgg tcagattcag tatctaagtc gataacttct
     5101 ggtttagacg aacccacctg tctcgtaggt ctggatgttg acgatcttac ggatttctta
     5161 tttttaccaa agaaatcaga aatattgacc atggagttca gcaggcgcag cagccagctc
     5221 aggctatcgc ccttgtcatc gtcatccttg tagtcgatgt catgatcttt ataatcaccg
     5281 tcatggtctt tgtagtcaga catggtatat ctccttatta aagttaaaca aaattatttc
     5341 tacaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcctaat
     5401 gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct
     5461 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
     5521 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
     5581 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
     5641 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
     5701 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
     5761 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
     5821 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
     5881 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
     5941 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
     6001 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
     6061 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
     6121 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
     6181 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
     6241 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
     6301 gcgttggtgc ggacatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
     6361 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     6421 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     6481 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     6541 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     6601 aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
     6661 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactaa catgagaatt
     6721 acaacttata tcgtatgggg ctgacttcag gtgctacatt tgaagagata aattgcactg
     6781 aaatctagag taacggaata gctgttcgtt gacttgatag accgattgat tcatcatctc
     6841 ataaataaag aaaaaccacc gctaccaacg gtggttttct caaggttcgc t
//
