LOCUS       sequence-214899-        7116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of SPAG1_TPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7116)
  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 7116)
  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_SPAG1_TPR
FEATURES             Location/Qualifiers
     source          1..7116
                     /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      1311..1358
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2951..3406)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3088..3109)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3300..3319
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3448..3470)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3556..3696
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3611..3630
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3711..3728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3882..4469)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4640..5431,5432..5500))
                     /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     5263..5282
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5501..5605)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5673..5691
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5714..5791
                     /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             5792..6874
                     /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(5811..5830)
                     /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 cggccgctac cggtcatccg ggcggtggcc agggtgcaga aaacccggcc ggtctgaaaa
      961 gtcagggcaa tgaactgttt cgttccggtc aattcgccga agcggccggc aagtattcag
     1021 cagctattgc actgctggaa ccggctggtt cggaaattgc ggatgacctg agcatcctgt
     1081 attctaaccg tgcggcctgc tacctgaaag aaggtaattg cagcggctgt atccaggact
     1141 gtaatcgcgc gctggaactg cacccgtttt ctatgaagcc gctgctgcgt cgcgcaatgg
     1201 cttatgaaac cctggaacaa tacggtaaag cctatgtgga ttacaagacg gttctgcaga
     1261 ttgactgcgg cctgcaactg gcaaatgatt ccgtcgcggc cgcataataa accgatacaa
     1321 ttaaaggctc cttttggagc cttttttttg gagattttca acgtgaaaaa attattattc
     1381 gcaattcctt tagttgttcc tttctattct cactccgctg aaactgttga aagttgttta
     1441 gcaaaacccc atacagaaaa ttcatttact aacgtctgga aagacgacaa aactttagat
     1501 cgttacgcta actatgaggg ctgtctgtgg aatgctacag gcgttgtagt ttgtactggt
     1561 gacgaaactc agtgtctagc tagagtggcg gtggctctgg ttccggtgat tttgattatg
     1621 aaaagatggc aaacgctaat aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac
     1681 agtctgacgc taaaggcaaa cttgattctg tcgctactga ttacggtgct gctatcgatg
     1741 gtttcattgg tgacgtttcc ggccttgcta atggtaatgg tgctactggt gattttgctg
     1801 gctctaattc ccaaatggct caagtcggtg acggtgataa ttcaccttta atgaataatt
     1861 tccgtcaata tttaccttcc ctccctcaat cggttgaatg tcgccctttt gtctttggcg
     1921 ctggtaaacc atatgaattt tctattgatt gtgacaaaat aaacttattc cgtggtgtct
     1981 ttgcgtttct tttatatgtt gccaccttta tgtatgtatt ttctacgttt gctaacatac
     2041 tgcgtaataa ggagtcttaa tcatgccagt tcttttggct agcgccgccc tataccttgt
     2101 ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc
     2161 cggcggcacc tcgctaacgg attcaccact ccaagaattg gagccaatca attcttgcgg
     2221 agaactgtga atgcgcaaac caacccttgg cagaacatat ccatcgcgtc cgccatctcc
     2281 agcagccgca cgcggcgcat ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc
     2341 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
     2401 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
     2461 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     2521 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     2581 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     2641 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     2701 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     2761 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     2821 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     2881 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     2941 aggatccgcg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     3001 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     3061 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     3121 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     3181 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     3241 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     3301 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     3361 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcgcg gatcctgcct
     3421 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3481 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3541 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg
     3601 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     3661 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact
     3721 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3781 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3841 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3901 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3961 taaagatacc agggtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4021 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4081 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4141 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4201 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4261 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4321 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4381 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4441 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4501 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4561 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4621 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4681 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4741 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4801 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4861 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4921 cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt
     4981 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5041 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5101 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5161 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5221 gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata
     5281 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5341 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5401 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5461 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5521 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5581 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     5641 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     5701 ttcaagaatt cccgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg
     5761 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     5821 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     5881 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     5941 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     6001 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     6061 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     6121 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     6181 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     6241 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     6301 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     6361 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     6421 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     6481 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     6541 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
     6601 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     6661 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     6721 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     6781 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     6841 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     6901 tcactcatta ggcacaattc tcatgtttga cagcttatca tcgactgcac ggtgcaccaa
     6961 tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac
     7021 tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca
     7081 tcataacggt tctggcaaat attctgaaat gagctg
//
