LOCUS       sequence-314733-        5636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses R2x2_VAL88 in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5636)
  AUTHORS   Koga R, Yamamoto M, Kosugi T, Kobayashi N, Sugiki T, Fujiwara T, 
            Koga N
  TITLE     Robust folding of a de novo designed ideal protein even with most of
            the core mutated to valine.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Nov 23. pii: 2002120117. doi: 
            10.1073/pnas.2002120117.
  PUBMED    33229587
REFERENCE   2  (bases 1 to 5636)
  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: "Proc Natl 
            Acad Sci U S A. 2020 Nov 23. pii: 2002120117. doi: 
            10.1073/pnas.2002120117."
COMMENT     Sequence Label: R2x2_VAL88
FEATURES             Location/Qualifiers
     source          1..5636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     39..58
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        39..57
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    58..82
                     /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)."
     RBS             97..119
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             406..423
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(476..494)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      490..537
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      574..1029
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(661..680)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     871..892
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1055..1159
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1160..1228,1229..2020)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1378..1397)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2191..2779
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2680..2699
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2933..2950
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2965..3104
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3030..3049)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3190..3212
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3206..3397)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4206..5288)
                     /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     5250..5269
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5289..5366)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5572..5591
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
       61 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
      121 atacatatgg gtcgcgtggt tgttgtggta acctctgaac aggtgaaaga agaagttcgt
      181 aagaaattcc cgcaagttga agtccgtgtg gttacgaccg aagaagacgc gaaacaggtt
      241 gtcaaggaag tccagaagaa aggtgtacag aaagttgtag tggttggtgt ttctgaaaaa
      301 gtggtgcaga aagtcaaaca ggaagcaaac gttcaggttt accgtgttac ctctaacgac
      361 gaagtggagc aggttgttaa agacgttaaa ggttctggtc tcgagcacca ccaccaccac
      421 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      481 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      541 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
      601 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      661 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      721 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      781 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
      841 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
      901 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
      961 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1021 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1081 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     1141 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     1201 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     1261 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     1321 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     1381 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     1441 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     1501 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     1561 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     1621 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     1681 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     1741 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     1801 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     1861 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     1921 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     1981 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     2041 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     2101 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2161 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2221 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2281 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     2341 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2401 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2461 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2521 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2581 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     2641 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2701 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     2761 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2821 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2881 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2941 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3001 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3061 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     3121 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3181 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3241 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     3301 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     3361 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     3421 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     3481 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     3541 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     3601 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     3661 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     3721 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     3781 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     3841 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     3901 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     3961 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     4021 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     4081 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     4141 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4201 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4261 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     4321 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     4381 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     4441 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     4501 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     4561 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     4621 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     4681 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     4741 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     4801 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     4861 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     4921 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     4981 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     5041 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     5101 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     5161 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     5221 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     5281 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     5341 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     5401 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     5461 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     5521 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     5581 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgc
//
