LOCUS       sequence-475695-        6543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpress Sc FLAG-S6-PCNA in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6543)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 6543)
  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-S6-PCNA-pET
FEATURES             Location/Qualifiers
     source          1..6543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    26..165
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(91..110)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     251..273
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(267..458)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2813..2832
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2852..2929)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3135..3154
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    3256..3280
                     /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)."
     RBS             3295..3317
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3327..3392
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4282..4300)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4296..4343
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4518..4523,4524..4540,4541..4546))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4573..4591)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4659..4763
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4764..4832,4833..5624)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4982..5001)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5795..6383
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6284..6303
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(6537..6543,1..11)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
       61 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
      121 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
      181 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
      241 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
      301 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
      361 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
      421 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
      481 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
      541 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
      601 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
      661 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
      721 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
      781 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
      841 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
      901 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
      961 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     1021 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     1081 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     1141 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     1201 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
     1261 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     1321 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     1381 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     1441 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     1501 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     1561 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     1621 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     1681 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     1741 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaactgtc
     1801 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     1861 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     1921 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     1981 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     2041 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     2101 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     2161 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     2221 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     2281 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     2341 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     2401 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     2461 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     2521 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     2581 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     2641 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     2701 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     2761 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     2821 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     2881 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     2941 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     3001 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     3061 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     3121 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     3181 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     3241 tacgactcat ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     3301 taactttaag aaggagatat accatggact acaaagacca tgacggtgat tataaagatc
     3361 atgacatcga ctacaaggat gacgatgaca agcgatccac tagtcatatc ggcgatagcc
     3421 tgagctggct gctgcgcctg ctgaaccata tgttagaagc aaaatttgaa gaagcatccc
     3481 ttttcaagag aataattgat ggtttcaaag attgtgtcca gttggtcaat ttccaatgta
     3541 aagaagatgg tatcattgca caagctgtcg atgactcaag agttctattg gtctccttgg
     3601 aaataggtgt cgaagccttc caagaatata gatgtgacca tcctgttacg ttaggtatgg
     3661 atctaacctc actaagtaaa atcctacgtt gtggtaacaa caccgataca ttaacactaa
     3721 ttgctgacaa cacaccggat tccatcatct tattatttga ggataccaag aaagaccgta
     3781 tagccgaata ctctctgaaa ttgatggata tcgatgctga tttcttaaag attgaagaat
     3841 tacagtacga ctccaccctg tcattgccat cttccgaatt ctctaaaatt gttcgtgact
     3901 tgtcccaatt gagtgattct attaatatca tgatcaccaa agaaacaata aagtttgtag
     3961 ctgacggtga tatcggatca ggttcagtca taataaaacc attcgtggat atggaacatc
     4021 ctgaaacaag catcaaactt gaaatggatc aacctgtcga cttgacgttc ggagctaaat
     4081 atttattgga catcattaag ggctcctccc tttctgatag agttggtatc aggctctcca
     4141 gcgaagctcc tgctttattc caatttgatt tgaagagtgg gttcctacag tttttcttgg
     4201 ctcctaaatt taatgacgaa gaataaagct tggatccggc tgctaacaaa gcccgaaagg
     4261 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     4321 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag
     4381 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     4441 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     4501 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt
     4561 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     4621 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4681 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4741 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     4801 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4861 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4921 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     4981 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     5041 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5101 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5161 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5221 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5281 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     5341 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     5401 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     5461 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     5521 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     5581 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     5641 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5701 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5761 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5821 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5881 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5941 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6001 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6061 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6121 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6181 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6241 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6301 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6361 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6421 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6481 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     6541 agt
//
