LOCUS       sequence-213877-        7026 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of PUF60_RRM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7026)
  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 7026)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_PUF60_RRM
FEATURES             Location/Qualifiers
     source          1..7026
                     /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      1221..1268
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2861..3316)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2998..3019)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3210..3229
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3358..3380)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3466..3606
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3521..3540
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3621..3638)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3792..4379)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4550..5341,5342..5410))
                     /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     5173..5192
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5411..5515)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5583..5601
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5624..5701
                     /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             5702..6784
                     /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(5721..5740)
                     /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 cggccgagtc taccgtaatg gttctgcgta acatggtgga tccgaaagat atcgacgacg
      961 acctggaagg cgaagttact gaagagtgcg gcaaattcgg tgccgtcaat cgcgtgatta
     1021 tctaccaaga aaagcagggt gaagaagaag acgcggagat catcgttaaa attttcgtgg
     1081 aattctccat tgcgagcgag acgcacaaag cgatccaggc tctgaacggt cgctggtttg
     1141 ctggccgtaa agtagttgca gaagtttatg accaggaacg tttcgataac tccgatctgt
     1201 ctgcagcggc cgcataataa accgatacaa ttaaaggctc cttttggagc cttttttttg
     1261 gagattttca acgtgaaaaa attattattc gcaattcctt tagttgttcc tttctattct
     1321 cactccgctg aaactgttga aagttgttta gcaaaacccc atacagaaaa ttcatttact
     1381 aacgtctgga aagacgacaa aactttagat cgttacgcta actatgaggg ctgtctgtgg
     1441 aatgctacag gcgttgtagt ttgtactggt gacgaaactc agtgtctagc tagagtggcg
     1501 gtggctctgg ttccggtgat tttgattatg aaaagatggc aaacgctaat aagggggcta
     1561 tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc taaaggcaaa cttgattctg
     1621 tcgctactga ttacggtgct gctatcgatg gtttcattgg tgacgtttcc ggccttgcta
     1681 atggtaatgg tgctactggt gattttgctg gctctaattc ccaaatggct caagtcggtg
     1741 acggtgataa ttcaccttta atgaataatt tccgtcaata tttaccttcc ctccctcaat
     1801 cggttgaatg tcgccctttt gtctttggcg ctggtaaacc atatgaattt tctattgatt
     1861 gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct tttatatgtt gccaccttta
     1921 tgtatgtatt ttctacgttt gctaacatac tgcgtaataa ggagtcttaa tcatgccagt
     1981 tcttttggct agcgccgccc tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg
     2041 gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg attcaccact
     2101 ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac caacccttgg
     2161 cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat ctcgggcagc
     2221 gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc
     2281 tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc
     2341 gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt
     2401 gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc
     2461 atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg
     2521 cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc
     2581 tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc
     2641 tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca
     2701 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta
     2761 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg
     2821 cttcacgacc acgctgatga gctttaccgc aggatccgcg aaattgtaag cgttaatatt
     2881 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2941 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     3001 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     3061 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     3121 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3181 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     3241 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3301 ccgctacagg gcgcgtcgcg gatcctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     3361 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     3421 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     3481 gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta
     3541 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     3601 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3661 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3721 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3781 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3841 agtcagaggt ggcgaaaccc gacaggacta taaagatacc agggtttccc cctggaagct
     3901 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3961 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4021 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4081 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4141 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4201 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     4261 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4321 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4381 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4441 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4501 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4561 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4621 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4681 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4741 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4801 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4861 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4921 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4981 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5041 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5101 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5161 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5221 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5281 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5341 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5401 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5461 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5521 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     5581 aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt cccgacacca tcgaatggtg
     5641 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     5701 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     5761 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     5821 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     5881 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     5941 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     6001 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     6061 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     6121 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     6181 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     6241 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     6301 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     6361 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     6421 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     6481 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     6541 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     6601 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     6661 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     6721 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     6781 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcacaattc tcatgtttga
     6841 cagcttatca tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc
     6901 tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc
     6961 ccgttctgga taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat
     7021 gagctg
//
