LOCUS       sequence-213646-        6933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of RPA2_RPA-C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6933)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 6933)
  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"; journalName: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_RPA2_RPA-C
FEATURES             Location/Qualifiers
     source          1..6933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      1128..1175
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2768..3223)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2905..2926)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3117..3136
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3265..3287)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3373..3513
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3428..3447
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3528..3545)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3699..4286)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4457..5248,5249..5317))
                     /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     5080..5099
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5318..5422)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5490..5508
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5531..5608
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             5609..6691
                     /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(5628..5647)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccgcaaa cggtctgacg gttgcgcaga accaagtact gaacctgatt aaggcgtgcc
      961 cacgtccgga gggcctgaat ttccaggatc tgaaaaacca gctgaaacac atgtctgtgt
     1021 cctctatcaa acaggccgtc gacttcctga gcaacgaagg tcacatctac tctactgttg
     1081 acgatgacca ttttaaatcc accgatgctg aagcggccgc ataataaacc gatacaatta
     1141 aaggctcctt ttggagcctt ttttttggag attttcaacg tgaaaaaatt attattcgca
     1201 attcctttag ttgttccttt ctattctcac tccgctgaaa ctgttgaaag ttgtttagca
     1261 aaaccccata cagaaaattc atttactaac gtctggaaag acgacaaaac tttagatcgt
     1321 tacgctaact atgagggctg tctgtggaat gctacaggcg ttgtagtttg tactggtgac
     1381 gaaactcagt gtctagctag agtggcggtg gctctggttc cggtgatttt gattatgaaa
     1441 agatggcaaa cgctaataag ggggctatga ccgaaaatgc cgatgaaaac gcgctacagt
     1501 ctgacgctaa aggcaaactt gattctgtcg ctactgatta cggtgctgct atcgatggtt
     1561 tcattggtga cgtttccggc cttgctaatg gtaatggtgc tactggtgat tttgctggct
     1621 ctaattccca aatggctcaa gtcggtgacg gtgataattc acctttaatg aataatttcc
     1681 gtcaatattt accttccctc cctcaatcgg ttgaatgtcg cccttttgtc tttggcgctg
     1741 gtaaaccata tgaattttct attgattgtg acaaaataaa cttattccgt ggtgtctttg
     1801 cgtttctttt atatgttgcc acctttatgt atgtattttc tacgtttgct aacatactgc
     1861 gtaataagga gtcttaatca tgccagttct tttggctagc gccgccctat accttgtctg
     1921 cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg
     1981 cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga
     2041 actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc
     2101 agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg
     2161 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     2221 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     2281 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     2341 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     2401 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     2461 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     2521 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     2581 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     2641 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     2701 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagg
     2761 atccgcgaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     2821 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     2881 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     2941 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     3001 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     3061 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     3121 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     3181 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcgcggat cctgcctcgc
     3241 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     3301 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     3361 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     3421 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     3481 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     3541 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3601 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3661 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3721 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3781 agataccagg gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3841 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3901 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3961 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4021 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4081 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     4141 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4201 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4261 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4321 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4381 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4441 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4501 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4561 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4621 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4681 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4741 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     4801 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4861 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4921 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4981 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5041 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     5101 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5161 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5221 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5281 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5341 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5401 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     5461 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     5521 aagaattccc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc
     5581 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     5641 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     5701 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     5761 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     5821 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     5881 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     5941 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     6001 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     6061 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     6121 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     6181 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     6241 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     6301 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     6361 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     6421 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     6481 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     6541 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     6601 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     6661 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     6721 ctcattaggc acaattctca tgtttgacag cttatcatcg actgcacggt gcaccaatgc
     6781 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
     6841 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
     6901 taacggttct ggcaaatatt ctgaaatgag ctg
//
