LOCUS       sequence-308008-        5817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3E2H.37 de novo designed protein in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5817)
  AUTHORS   Yang C, Sesterhenn F, Bonet J, van Aalen EA, Scheller L, Abriata LA,
            Cramer JT, Wen X, Rosset S, Georgeon S, Jardetzky T, Krey T, 
            Fussenegger M, Merkx M, Correia BE
  TITLE     Bottom-up de novo design of functional proteins with complex 
            structural features.
  JOURNAL   Nat Chem Biol. 2021 Apr;17(4):492-500. doi: 
            10.1038/s41589-020-00699-x. Epub 2021 Jan 4.
  PUBMED    33398169
REFERENCE   2  (bases 1 to 5817)
  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"; doi: 
            "10.1038/s41589-020-00699-x"; journalName: "Nat Chem Biol"; date: 
            "2021-04"; volume: "17"; issue: "4"; pages: "492-500"
COMMENT     Sequence Label: pET_3E2H.37
FEATURES             Location/Qualifiers
     source          1..5817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(19..37)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        105..209
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(210..278,279..1070)
                     /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     complement(428..447)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1241..1829
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1730..1749
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1983..2000
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2015..2154
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2080..2099)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2240..2262
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2256..2447)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3759..4841)
                     /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     4803..4822
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4842..4919)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5125..5144
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5228..5247
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5228..5246
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5247..5271
                     /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             5286..5308
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5325..5342
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5559..5606
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5781..5786,5787..5803,5804..5809))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
       61 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
      121 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
      181 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
      241 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
      301 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
      361 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
      421 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
      481 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
      541 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
      601 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
      661 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
      721 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
      781 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
      841 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
      901 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
      961 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     1021 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     1081 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     1141 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     1201 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     1261 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     1321 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     1381 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     1441 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     1501 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     1561 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     1621 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     1681 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     1741 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     1801 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     1861 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     1921 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     1981 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     2041 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     2101 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     2161 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     2221 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     2281 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     2341 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     2401 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     2461 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     2521 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     2581 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     2641 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     2701 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     2761 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     2821 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     2881 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg
     2941 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt
     3001 tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg
     3061 ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
     3121 gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
     3181 atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta
     3241 ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc
     3301 tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa
     3361 tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg
     3421 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     3481 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     3541 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     3601 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     3661 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     3721 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3781 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3841 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     3901 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     3961 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     4021 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     4081 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     4141 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     4201 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     4261 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     4321 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     4381 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     4441 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     4501 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     4561 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     4621 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     4681 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     4741 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     4801 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     4861 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     4921 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     4981 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     5041 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     5101 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     5161 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     5221 gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa
     5281 ataattttgt ttaactttaa gaaggagata tacatatggg tggtcaccat caccaccatc
     5341 attggtccgg tccgacctgc aaaatcaccg ttcgtctgaa cgaccgtggt atcatcaaaa
     5401 ccgttaacac catctctggt ggttctcgtc gtgcgctgga agcgatcgaa gaagcgcgtc
     5461 aggcgggtat ggacgttacc ttcgactgcg gtggtgaact gcgtgaacag ctggaaaaag
     5521 ttcaggaagt tgttgaaaaa taataggctg agcaataact agcataaccc cttggggcct
     5581 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa
     5641 gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc
     5701 gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt
     5761 taactgtgat aaactaccgc attaaagctt atcgatgata agctgtcaaa catgaga
//
