LOCUS       sequence-482405-        5757 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Over-express FLAG-S6-rad24 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5757)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 5757)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: Sc FLAG-S6-rad24-pRSFDuet
FEATURES             Location/Qualifiers
     source          1..5757
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1247..1996
                     /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."
     primer_bind     1727..1746
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1969..1986
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        2012..2103
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2104..2919
                     /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             2104..2919
                     /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(2177..2196)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(3149..3196)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3192..3210
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3248..3292)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(5447..5512)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    5559..5583
                     /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(5583..5602)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5584..5602)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        5648..5725
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5726..5757,1..1051)
                     /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(join(5745..5757,1..7))
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
       61 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
      121 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
      181 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
      241 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
      301 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
      361 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
      421 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
      481 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
      541 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
      601 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
      661 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
      721 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggaca tctcggtagt
      781 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
      841 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
      901 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
      961 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     1021 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     1081 ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
     1141 tccggtgggc gcggggcatg actaacatga gaattacaac ttatatcgta tggggctgac
     1201 ttcaggtgct acatttgaag agataaattg cactgaaatc tagagtaacg gaatagctgt
     1261 tcgttgactt gatagaccga ttgattcatc atctcataaa taaagaaaaa ccaccgctac
     1321 caacggtggt tttctcaagg ttcgctgagc taccaactct ttgaaccaag gtaagtgggt
     1381 tggaggaccg cactcaccaa aatctgttct ttcagtttag ccttaacagg tgcataactt
     1441 caagacaaag tcctctaaat cagttaccaa tggctgctgc cagtggcgat aagtcgtgtc
     1501 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     1561 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agataccaac
     1621 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     1681 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     1741 atctttatag tcctgtcggg tttcgccacc tctggcttga gcgtcgattt ttgtgatgct
     1801 cgtcaggggg gcggagccta tggaaaaacg cctgcggcgt tggcttcttc cggtgctttg
     1861 ctttttgctc acatgttctt tccggcttta tcccctgatt ctgtggataa ccgtattacc
     1921 gcttttgagt gagctgacac cgctcgccgc agtcgaacga ccgagcgtag cgagtcagtg
     1981 agcgaggaag cggaagagcg ctgcatgcct atttgtttat ttttctaaat acattcaaat
     2041 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2101 agtatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg
     2161 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     2221 atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     2281 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     2341 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     2401 gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     2461 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     2521 ccttttaaca gcgaccgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     2581 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     2641 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     2701 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     2761 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     2821 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     2881 aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata
     2941 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     3001 agtgccactt gcggagaccc ggtcgtcagc ttgtcgtcgg ttcagggcag ggtcgttaaa
     3061 tagccgctta tgtctattgc tggtttaccg gtttattgac taccggaagc agtgtgaccg
     3121 tgtgcttctc aaatgcctga ggtttcagca aaaaacccct caagacccgt ttagaggccc
     3181 caaggggtta tgctagttat tgctcagcgg tggcagcagc ctaggttaat taagctgcgc
     3241 tagtagacga gtccatgtgc tggcgttcaa atttcgcagc agcggtttct ttaccagact
     3301 cgagggtacc gacgtcagcg atcgcgtggc cggccgatat ccaattgaga tcatagattt
     3361 gtgtggaata tttcctgggg ttttctcgtc aaatttaaag agtaaaaagt tagagtattt
     3421 ccagatctga atctgaaagg gactcactga taactggcgc tttacgcggc tgtctctggg
     3481 acagaatttc acatagagac tcgtcttcgt cgcttctacc aaatgtttca ttgccaatat
     3541 tattgctgtt atcgctatcg ctatcgacaa tagggtcatc attaaaagaa gtttcttcgt
     3601 cttcaagatc gtcgttagcc atcatcacat ttctttcata ttggtcaatc aacatgcgaa
     3661 gccttctgtc cctttccttt ttttgatcct ctaaatggtc acaattattg ctatcattat
     3721 ccatcaatgg tgctagtcca tcctccacag atagtgcttc gataggcccg cctatccgat
     3781 ccacaacgtc gattccgttc tcaactttca ttatatcact aaatttgtcc atggtttgtt
     3841 tgggccccga ggagccactc ggaagattct tcaaataata gagaatgtat ttttttttat
     3901 aactctgaca ctttctaatt agaggtgcgt agtagccaaa ttctagagtt atattcctga
     3961 aagaatgtac cgcgttgtac ttataaagac taacatttaa ccaatcttca gcttgaactt
     4021 taaatgaatt ttgtaatttt cttactttcc attctcttgg aaaataaacc gttccatgat
     4081 tatggccttg tttactgatg ttacgaaagg ttttgcgcac ttctcgtaaa ccatactcat
     4141 tggattctgg tagaccattc atattatcac actcgctcag gcaatccaca attgatgatg
     4201 catcagaaat gctgaattcg cctttattaa atgtgttata gttctctaat attcctaatt
     4261 tgaaatcctc tttcgataac aaattgttcg aattttcgaa gaggttatta atcatttcgt
     4321 tatcgttatt cgtgctatgg gaaccatgta tcaccttccc aatggcatga aagtatgata
     4381 tggtggattc tcgggttgaa atcggcaaac ttccacttga tgtcgcccaa aattgaaggg
     4441 tcgtaatggc cgacctaata tcaccagtct cttgcgcaat ataatctatg acttcctgtc
     4501 ttttattcca tttatttttc tccttcaaca ttttcatatt ctgtacacaa ataaatttca
     4561 agtgcttttt taataaagtg ctgttaattg gattaaactt aattcttttc aaccttggat
     4621 gcatcaatat ttctttgttc attatggttt ctgcactaaa tgtataatca ataccaaatt
     4681 tgcgataatt attatcgttc tctggaattt cacattcagt tatacatata acaaggggag
     4741 gtaatagagg ctccgaacta tatagccact gtaatataag ttgttgaaat cgacgtctgg
     4801 tatctatatg gaagacgttg ggaaggtcct cgataagtat taatgacagg ttggacatca
     4861 caagataccg tgcgcctttt aagaactcac taaagctttc catctgagga agatcgttga
     4921 ctatacaatc acctctaaac tcggtcactt tatgctcgtt cggggtgctt cgaaaggacg
     4981 ttccgttgct gttttgtctg tatttaggaa ctaagatttt tgagagttct tttatgaccg
     5041 tacttttaga gcatccactg gggccagaca gtagtaggat cctatgcttg gcgttaggta
     5101 aaaacatggc atctaaagct tcttgtacat ctttaagttt tcttttatgt atggccactt
     5161 gctccaaaca attgggcttg aacttttcgt accactgttc accgccgtcg tcgttgatac
     5221 ttgggaggat actagacgta tcttgcttgg aaagaaagtc attttcagtg cttctcgcct
     5281 tacgaactgg cgaagtcggt ctagatgagc tccattttgt tatttgcgag cccaatgaac
     5341 tgagactata ttgtaataag ggccgtttgt tcaaattcgt actatccata tggttcagca
     5401 ggcgcagcag ccagctcagg ctatcgccga tatgactagt ggatcgcttg tcatcgtcat
     5461 ccttgtagtc gatgtcatga tctttataat caccgtcatg gtctttgtag tccatggtat
     5521 atctccttat taaagttaaa caaaattatt tctacagggg aattgttatc cgctcacaat
     5581 tcccctatag tgagtcgtat taatttccta atgcaggagt cgcataaggg agagcgtcga
     5641 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     5701 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcaga
//
