LOCUS       sequence-215044-        7077 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of SPRED3_WH1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7077)
  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 7077)
  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_SPRED3_WH1
FEATURES             Location/Qualifiers
     source          1..7077
                     /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      1272..1319
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2912..3367)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3049..3070)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3261..3280
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3409..3431)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3517..3657
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3572..3591
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3672..3689)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3843..4430)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4601..5392,5393..5461))
                     /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     5224..5243
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5462..5566)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5634..5652
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5675..5752
                     /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             5753..6835
                     /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(5772..5791)
                     /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 cggccatggt tcgtgtccgt gctgttgtga tggctcgtga tgattcgtca ggcggttggc
      961 tgccggttgg cggtggtggt ctgtcgcaag tcagcgtgtg ccgtgttcgt ggtgcacgtc
     1021 cggaaggcgg tgcacgtcag ggccattatg ttattcacgg tgaacgtctg cgcgatcaaa
     1081 aaaccacgct ggaatgcacc ctgaaaccgg gtctggtcta caacaaagtg aatccgatct
     1141 ttcatcactg gagtctgggc gattgtaaat ttggtctgac gttccagagc ccggcggaag
     1201 ccgacgaatt ccaaaagtct ctgctggcgg ccctggcagc tctgggccgt ggttccgcgg
     1261 ccgcataata aaccgataca attaaaggct ccttttggag cctttttttt ggagattttc
     1321 aacgtgaaaa aattattatt cgcaattcct ttagttgttc ctttctattc tcactccgct
     1381 gaaactgttg aaagttgttt agcaaaaccc catacagaaa attcatttac taacgtctgg
     1441 aaagacgaca aaactttaga tcgttacgct aactatgagg gctgtctgtg gaatgctaca
     1501 ggcgttgtag tttgtactgg tgacgaaact cagtgtctag ctagagtggc ggtggctctg
     1561 gttccggtga ttttgattat gaaaagatgg caaacgctaa taagggggct atgaccgaaa
     1621 atgccgatga aaacgcgcta cagtctgacg ctaaaggcaa acttgattct gtcgctactg
     1681 attacggtgc tgctatcgat ggtttcattg gtgacgtttc cggccttgct aatggtaatg
     1741 gtgctactgg tgattttgct ggctctaatt cccaaatggc tcaagtcggt gacggtgata
     1801 attcaccttt aatgaataat ttccgtcaat atttaccttc cctccctcaa tcggttgaat
     1861 gtcgcccttt tgtctttggc gctggtaaac catatgaatt ttctattgat tgtgacaaaa
     1921 taaacttatt ccgtggtgtc tttgcgtttc ttttatatgt tgccaccttt atgtatgtat
     1981 tttctacgtt tgctaacata ctgcgtaata aggagtctta atcatgccag ttcttttggc
     2041 tagcgccgcc ctataccttg tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc
     2101 cacctcgacc tgaatggaag ccggcggcac ctcgctaacg gattcaccac tccaagaatt
     2161 ggagccaatc aattcttgcg gagaactgtg aatgcgcaaa ccaacccttg gcagaacata
     2221 tccatcgcgt ccgccatctc cagcagccgc acgcggcgca tctcgggcag cgttgggtcc
     2281 tggccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt
     2341 tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc
     2401 tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa
     2461 agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga
     2521 tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc
     2581 tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt
     2641 tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc
     2701 atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa
     2761 caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg
     2821 gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac
     2881 cacgctgatg agctttaccg caggatccgc gaaattgtaa gcgttaatat tttgttaaaa
     2941 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     3001 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     3061 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3121 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     3181 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3241 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3301 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3361 ggcgcgtcgc ggatcctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     3421 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3481 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     3541 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     3601 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     3661 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3721 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3781 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3841 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3901 tggcgaaacc cgacaggact ataaagatac cagggtttcc ccctggaagc tccctcgtgc
     3961 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4021 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4081 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4141 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4201 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4261 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4321 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4381 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4441 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4501 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4561 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4621 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4681 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4741 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4801 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4861 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag
     4921 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4981 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5041 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5101 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5161 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac
     5221 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5281 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5341 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5401 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5461 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5521 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5581 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5641 gtatcacgag gccctttcgt cttcaagaat tcccgacacc atcgaatggt gcaaaacctt
     5701 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
     5761 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     5821 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     5881 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     5941 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     6001 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     6061 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     6121 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     6181 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     6241 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     6301 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     6361 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     6421 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     6481 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     6541 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     6601 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     6661 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     6721 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6781 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     6841 acgcaattaa tgtaagttag ctcactcatt aggcacaatt ctcatgtttg acagcttatc
     6901 atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg
     6961 gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg
     7021 ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctg
//
