LOCUS       sequence-215947-        7086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of RPA1_RepA-N.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7086)
  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 7086)
  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_RPA1_RepA-N
FEATURES             Location/Qualifiers
     source          1..7086
                     /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      1281..1328
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2921..3376)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3058..3079)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3270..3289
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3418..3440)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3526..3666
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3581..3600
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3681..3698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3852..4439)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4610..5401,5402..5470))
                     /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     5233..5252
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5471..5575)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5643..5661
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5684..5761
                     /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             5762..6844
                     /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(5781..5800)
                     /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 cggccgtcgg ccagctgagc gaaggtgcga ttgcggccat catgcaaaaa ggcgatacca
      961 atattaaacc gatcctgcag gttattaaca tccgtccgat taccacgggc aattcaccgc
     1021 cgcgttatcg cctgctgatg tcggacggtc tgaacaccct gagctctttt atgctggcaa
     1081 cgcaactgaa cccgctggtt gaagaagaac agctgagttc caattgcgtt tgtcaaattc
     1141 atcgcttcat cgtcaacacg ctgaaagatg gccgtcgcgt ggttattctg atggaactgg
     1201 aagtgctgaa aagtgcagaa gctgtcggcg tgaaaatcgg taacccggtt ccgtacaatg
     1261 aaggtgcggc cgcataataa accgatacaa ttaaaggctc cttttggagc cttttttttg
     1321 gagattttca acgtgaaaaa attattattc gcaattcctt tagttgttcc tttctattct
     1381 cactccgctg aaactgttga aagttgttta gcaaaacccc atacagaaaa ttcatttact
     1441 aacgtctgga aagacgacaa aactttagat cgttacgcta actatgaggg ctgtctgtgg
     1501 aatgctacag gcgttgtagt ttgtactggt gacgaaactc agtgtctagc tagagtggcg
     1561 gtggctctgg ttccggtgat tttgattatg aaaagatggc aaacgctaat aagggggcta
     1621 tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc taaaggcaaa cttgattctg
     1681 tcgctactga ttacggtgct gctatcgatg gtttcattgg tgacgtttcc ggccttgcta
     1741 atggtaatgg tgctactggt gattttgctg gctctaattc ccaaatggct caagtcggtg
     1801 acggtgataa ttcaccttta atgaataatt tccgtcaata tttaccttcc ctccctcaat
     1861 cggttgaatg tcgccctttt gtctttggcg ctggtaaacc atatgaattt tctattgatt
     1921 gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct tttatatgtt gccaccttta
     1981 tgtatgtatt ttctacgttt gctaacatac tgcgtaataa ggagtcttaa tcatgccagt
     2041 tcttttggct agcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg
     2101 gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact
     2161 ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttgg
     2221 cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat ctcgggcagc
     2281 gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc
     2341 tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc
     2401 gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt
     2461 gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc
     2521 atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg
     2581 cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc
     2641 tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc
     2701 tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca
     2761 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta
     2821 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg
     2881 cttcacgacc acgctgatga gctttaccgc aggatccgcg aaattgtaag cgttaatatt
     2941 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     3001 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     3061 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     3121 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     3181 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3241 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     3301 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3361 ccgctacagg gcgcgtcgcg gatcctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     3421 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     3481 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     3541 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     3601 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     3661 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3721 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3781 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3841 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3901 agtcagaggt ggcgaaaccc gacaggacta taaagatacc agggtttccc cctggaagct
     3961 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4021 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4081 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4141 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4201 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4261 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     4321 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4381 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4441 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4501 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4561 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4621 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4681 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4741 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4801 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4861 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4921 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4981 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5041 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5101 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5161 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5221 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5281 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5341 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5401 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5461 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5521 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5581 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     5641 aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt cccgacacca tcgaatggtg
     5701 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     5761 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     5821 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     5881 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     5941 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     6001 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     6061 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     6121 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     6181 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     6241 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     6301 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     6361 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     6421 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     6481 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     6541 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     6601 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     6661 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     6721 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     6781 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     6841 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcacaattc tcatgtttga
     6901 cagcttatca tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc
     6961 tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc
     7021 ccgttctgga taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat
     7081 gagctg
//
