LOCUS       sequence-310168-        5865 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 4H.01 de novo designed protein in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5865)
  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 5865)
  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_4H.01
FEATURES             Location/Qualifiers
     source          1..5865
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1390..1409
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1390..1408
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1409..1433
                     /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             1448..1470
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1733..1750
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1769..1816
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1991..1996,1997..2013,2014..2019))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2046..2064)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2132..2236
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2237..2305,2306..3097)
                     /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(2455..2474)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3268..3856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3757..3776
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4010..4027
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4042..4181
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4107..4126)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4267..4289
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4283..4474)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5786..5865,1..1003))
                     /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"
ORIGIN
        1 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     1381 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1441 aaataatttt gtttaacttt aagaaggaga tatacatatg gaagttgaac gtgaactgcg
     1501 taactggctg tctgaagttc tgtctaaaat caacgacgcg ccggttacca acgacatcaa
     1561 aaaagcgatc tctaaccagg ttctgaaagt tgcggaacag gtttggaacg gtcactctaa
     1621 agaagaactg caggaacgtg ttcgtaaaga agtttgctct gtttgctcta acgttccggc
     1681 gtgctgggcg atctgcggtg gtctgctgga agttgttaaa taccagggct cgcaccacca
     1741 ccaccaccac taataggctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1801 cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa gaggcccggc
     1861 agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg
     1921 atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat
     1981 aaactaccgc attaaagctt atcgatgata agctgtcaaa catgagaatt cttgaagacg
     2041 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     2101 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2161 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     2221 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     2281 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     2341 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     2401 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     2461 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     2521 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     2581 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     2641 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     2701 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     2761 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     2821 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2881 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2941 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3001 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3061 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     3121 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     3181 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3241 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3301 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3361 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3421 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3481 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3541 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3601 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3661 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3721 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3781 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3841 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3901 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3961 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4021 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4081 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4141 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     4201 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4261 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     4321 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     4381 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     4441 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4501 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4561 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4621 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4681 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4741 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4801 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4861 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     4921 gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc
     4981 tgctggagat ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag
     5041 ttctccgcaa gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc
     5101 gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag
     5161 gcagacaagg tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga
     5221 ggcggcataa atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc
     5281 cgcgagcgat ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc
     5341 ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat
     5401 ccagcctcgc gtcgcgaacg ccagcaagac gtagcccagc gcgtcggccg ccatgccggc
     5461 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     5521 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     5581 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     5641 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     5701 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     5761 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5821 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggttt
//
