LOCUS       sequence-474606-        4948 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpress Sc RFC5 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4948)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 4948)
  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 RFC5-pCDFDuet
FEATURES             Location/Qualifiers
     source          1..4948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     576..595
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(615..692)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        738..756
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    757..781
                     /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)."
     CDS             837..854
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        968..986
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    987..1011
                     /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)."
     CDS             2222..2266
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2304..2322)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2318..2365
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2536..3327)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(3328..3419)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(join(4480..4948,1..614))
                     /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"
ORIGIN
        1 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
       61 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
      121 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
      181 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
      241 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
      301 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
      361 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
      421 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
      481 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
      541 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
      601 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
      661 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
      721 ctcctgcatt aggaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
      781 ccctgtagaa ataattttgt ttaactttaa taaggagata taccatgggc agcagccatc
      841 accatcatca ccacagccag gatccgaatt cgagctcggc gcgcctgcag gtcgacaagc
      901 ttgcggccgc ataatgctta agtcgaacag aaagtaatcg tattgtacac ggccgcataa
      961 tcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc cccatcttag
     1021 tatattagtt aagtataaga aggagatata catatgtcat tgtgggtaga taaatacaga
     1081 cctaagtcct tgaatgctct ttcacataat gaagagttga caaattttct aaaatcgtta
     1141 tctgatcagc ctcgtgattt acctcatctt ttactgtatg gaccaaatgg tacaggtaag
     1201 aaaacgcgtt gtatggcatt attggagtcc atatttggac ctggagtcta tagattgaaa
     1261 attgatgtca gacaatttgt cactgcttcg aacagaaaac tagaactgaa tgtggtcagc
     1321 tcgccatacc atttagagat cacgccaagt gatatgggta acaatgatag aattgtcatc
     1381 caagaactat tgaaagaagt ggctcaaatg gaacaagtgg actttcaaga ttctaaggat
     1441 ggacttgccc atagatataa gtgtgttatt atcaacgagg cgaactcgtt aacaaaagat
     1501 gctcaagctg ctttaagacg taccatggaa aaatactcca aaaacattag gttgataatg
     1561 gtctgcgatt cgatgtcgcc tataattgct cctatcaaat cccgttgtct gttgattcgt
     1621 tgtcctgcac caagcgatag cgaaatttca actatcttgt ctgatgtggt gacaaatgaa
     1681 agaatacaac tagaaacaaa ggatatttta aaaagaattg ctcaggcatc gaatggaaac
     1741 ttgcgagtct ccctattaat gcttgaatct atggcactga acaacgaatt agcattgaaa
     1801 agcagtagcc ctataataaa accagattgg attatagtga tccataaatt aacgaggaaa
     1861 atcgttaaag agagatctgt caattcttta atcgaatgca gagctgtcct atacgattta
     1921 ctagctcatt gtatacctgc caatatcatc ttaaaggaac taacgttttc tttgttggat
     1981 gtggaaaccc tgaataccac gaataaatcg tccataattg aatactcaag tgtttttgac
     2041 gaaagattat cacttggaaa caaagcaata ttccatttgg aagggttcat agcaaaagtt
     2101 atgtgctgtc tagattaatg taagatatgt cataaatact gtataagtca cacaaaaagc
     2161 ggatctcaat tggatatcgg ccggccacgc gatcgctgac gtcggtaccc tcgagtctgg
     2221 taaagaaacc gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc
     2281 ttaattaacc taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc
     2341 taaacgggtc ttgaggggtt ttttgctgaa acctcaggca tttgagaagc acacggtcac
     2401 actgcttccg gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg
     2461 accctgccct gaaccgacga ccgggtcatc gtggccggat cttgcggccc ctcggcttga
     2521 acgaattgtt agacattatt tgccgactac cttggtgatc tcgcctttca cgtagtggac
     2581 aaattcttcc aactgatctg cgcgcgaggc caagcgatct tcttcttgtc caagataagc
     2641 ctgtctagct tcaagtatga cgggctgata ctgggccggc aggcgctcca ttgcccagtc
     2701 ggcagcgaca tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa
     2761 cgtaagcact acatttcgct catcgccagc ccagtcgggc ggcgagttcc atagcgttaa
     2821 ggtttcattt agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga gttcctccgc
     2881 cgctggacct accaaggcaa cgctatgttc tcttgctttt gtcagcaaga tagccagatc
     2941 aatgtcgatc gtggctggct cgaagatacc tgcaagaatg tcattgcgct gccattctcc
     3001 aaattgcagt tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt gcacaacaat
     3061 ggtgacttct acagcgcgga gaatctcgct ctctccaggg gaagccgaag tttccaaaag
     3121 gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg taaccagcaa
     3181 atcaatatca ctgtgtggct tcaggccgcc atccactgcg gagccgtaca aatgtacggc
     3241 cagcaacgtc ggttcgagat ggcgctcgat gacgccaact acctctgata gttgagtcga
     3301 tacttcggcg atcaccgctt ccctcatact cttccttttt caatattatt gaagcattta
     3361 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3421 agctagctca ctcggtcgct acgctccggg cgtgagactg cggcgggcgc tgcggacaca
     3481 tacaaagtta cccacagatt ccgtggataa gcaggggact aacatgtgag gcaaaacagc
     3541 agggccgcgc cggtggcgtt tttccatagg ctccgccctc ctgccagagt tcacataaac
     3601 agacgctttt ccggtgcatc tgtgggagcc gtgaggctca accatgaatc tgacagtacg
     3661 ggcgaaaccc gacaggactt aaagatcccc accgtttccg gcgggtcgct ccctcttgcg
     3721 ctctcctgtt ccgaccctgc cgtttaccgg atacctgttc cgcctttctc ccttacggga
     3781 agtgtggcgc tttctcatag ctcacacact ggtatctcgg ctcggtgtag gtcgttcgct
     3841 ccaagctggg ctgtaagcaa gaactccccg ttcagcccga ctgctgcgcc ttatccggta
     3901 actgttcact tgagtccaac ccggaaaagc acggtaaaac gccactggca gcagccattg
     3961 gtaactggga gttcgcagag gatttgttta gctaaacacg cggttgctct tgaagtgtgc
     4021 gccaaagtcc ggctacactg gaaggacaga tttggttgct gtgctctgcg aaagccagtt
     4081 accacggtta agcagttccc caactgactt aaccttcgat caaaccacct ccccaggtgg
     4141 ttttttcgaa ggttaagtca gttggggaac tgcttaacct tcgatcaaac cacctcccca
     4201 ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga aaaaaaggat ctcaagaaga
     4261 tcctttgatc ttttctactg aaccgctcta gatttcagtg caatttatct cttcaaatgt
     4321 agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc
     4381 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     4441 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4501 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4561 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     4621 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     4681 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     4741 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     4801 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     4861 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     4921 cgctatcggc tgaatttgat tgcgagtg
//
