LOCUS       sequence-308009-        5871 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 4E2H.210 de novo protein in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5871)
  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 5871)
  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_4E2H.210
FEATURES             Location/Qualifiers
     source          1..5871
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..49)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        117..221
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(222..290,291..1082)
                     /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(440..459)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1253..1841
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1742..1761
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1995..2012
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2027..2166
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2092..2111)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2252..2274
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2268..2459)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3771..4853)
                     /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     4815..4834
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4854..4931)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5137..5156
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5240..5259
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5240..5258
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5259..5283
                     /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             5298..5320
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5337..5354
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5625..5672
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5847..5852,5853..5869,5870..5871,1..4))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
       61 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
      121 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
      181 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
      241 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
      301 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
      361 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
      421 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
      481 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
      541 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
      601 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
      661 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
      721 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
      781 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
      841 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
      901 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
      961 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     1021 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     1081 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     1141 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     1201 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     1261 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     1321 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     1381 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     1441 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     1501 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     1561 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     1621 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     1681 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     1741 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     1801 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     1861 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     1921 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     1981 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     2041 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     2101 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     2161 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     2221 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     2281 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     2341 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     2401 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     2461 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     2521 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     2581 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     2641 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     2701 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     2761 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     2821 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     2881 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg
     2941 ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc
     3001 caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga
     3061 gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc
     3121 atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg
     3181 ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa
     3241 tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt
     3301 catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga
     3361 gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc
     3421 ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     3481 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     3541 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     3601 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     3661 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     3721 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     3781 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3841 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     3901 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     3961 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     4021 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     4081 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     4141 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     4201 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     4261 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     4321 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     4381 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     4441 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     4501 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     4561 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     4621 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     4681 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     4741 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     4801 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     4861 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     4921 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     4981 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     5041 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     5101 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     5161 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     5221 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
     5281 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg gctagccacc
     5341 atcaccacca tcatggttcc ccgaacacca tctcttgcac cgcgccgacc cgtgacgaac
     5401 tggcgcagtg cgttcaggaa cactggaaag aatgggcgaa aaaactggac ggttctaaag
     5461 ttaccttctc tgcgaacgtt ccgaaactgt ctcgtggtat catcaaaacc tctgacggta
     5521 acatcgaaga agttctgcag gcgctggcga aagcgctgaa agaaggtgtt tctgttgaag
     5581 gtggtaccgt taccatctct ctgaaataat aggctgagca ataactagca taaccccttg
     5641 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc
     5701 ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac
     5761 ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta
     5821 gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct g
//
