LOCUS       sequence-474410-        6420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpress Sc PCNA in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6420)
  TITLE     O'Donnell lab plasmids 2025
REFERENCE   2  (bases 1 to 6420)
  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 PCNA-pET(11a)
FEATURES             Location/Qualifiers
     source          1..6420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      531..1119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1020..1039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1273..1290
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1305..1444
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1370..1389)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1530..1552
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1546..1737)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3049..4131)
                     /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     4093..4112
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4132..4209)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4415..4434
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4518..4537
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4518..4536
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4537..4561
                     /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             4576..4598
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(5438..5456)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5452..5499
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5674..5679,5680..5696,5697..5702))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5729..5747)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5815..5919
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5920..5988,5989..6420,1..360)
                     /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(6138..6157)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
       61 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
      121 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
      181 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
      241 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
      301 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
      361 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
      421 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
      481 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      541 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      601 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      661 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
      721 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
      781 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
      841 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
      901 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
      961 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1021 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1081 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1141 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     1201 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     1261 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     1321 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     1381 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     1441 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     1501 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     1561 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     1621 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     1681 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     1741 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     1801 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     1861 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     1921 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     1981 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     2041 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     2101 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     2161 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct
     2221 gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca
     2281 agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt
     2341 ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat
     2401 gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc
     2461 aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg
     2521 atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca
     2581 tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga
     2641 agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc
     2701 agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     2761 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     2821 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     2881 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     2941 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     3001 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     3061 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3121 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     3181 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     3241 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     3301 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     3361 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     3421 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     3481 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     3541 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     3601 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     3661 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     3721 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     3781 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     3841 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     3901 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     3961 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     4021 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     4081 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     4141 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     4201 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     4261 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     4321 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     4381 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     4441 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     4501 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     4561 ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgtt agaagcaaaa
     4621 tttgaagaag catccctttt caagagaata attgatggtt tcaaagattg tgtccagttg
     4681 gtcaatttcc aatgtaaaga agatggtatc attgcacaag ctgtcgatga ctcaagagtt
     4741 ctattggtct ccttggaaat aggtgtcgaa gccttccaag aatatagatg tgaccatcct
     4801 gttacgttag gtatggatct aacctcacta agtaaaatcc tacgttgtgg taacaacacc
     4861 gatacattaa cactaattgc tgacaacaca ccggattcca tcatcttatt atttgaggat
     4921 accaagaaag accgtatagc cgaatactct ctgaaattga tggatatcga tgctgatttc
     4981 ttaaagattg aagaattaca gtacgactcc accctgtcat tgccatcttc cgaattctct
     5041 aaaattgttc gtgacttgtc ccaattgagt gattctatta atatcatgat caccaaagaa
     5101 acaataaagt ttgtagctga cggtgatatc ggatcaggtt cagtcataat aaaaccattc
     5161 gtggatatgg aacatcctga aacaagcatc aaacttgaaa tggatcaacc tgtcgacttg
     5221 acgttcggag ctaaatattt attggacatc attaagggct cctccctttc tgatagagtt
     5281 ggtatcaggc tctccagcga agctcctgct ttattccaat ttgatttgaa gagtgggttc
     5341 ctacagtttt tcttggctcc taaatttaat gacgaagaat aaagcttgga tccggctgct
     5401 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     5461 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     5521 ggatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca
     5581 gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac
     5641 tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc
     5701 aaacatgaga attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5761 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5821 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5881 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5941 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6001 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6061 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6121 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     6181 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6241 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6301 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6361 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
//
