LOCUS       sequence-310182-        5507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 4E1H.95 de novo protein in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5507)
  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 5507)
  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_4E1H.95
FEATURES             Location/Qualifiers
     source          1..5507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(88..107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /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(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2360..2377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2392..2531
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2457..2476)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2617..2639
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2633..2824)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3633..4715)
                     /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     4677..4696
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4716..4793)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4999..5018
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5102..5121
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5102..5120
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5121..5145
                     /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             5160..5182
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5388..5405
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5424..5471
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
       61 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      181 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2341 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2401 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
     2461 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     2521 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     2581 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     2641 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     2701 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     2761 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     2821 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     2881 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     2941 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     3001 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     3061 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     3121 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     3181 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     3241 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     3301 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     3361 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     3421 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     3481 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     3541 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     3601 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3661 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3721 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     3781 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     3841 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     3901 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     3961 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     4021 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     4081 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     4141 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     4201 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     4261 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     4321 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     4381 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     4441 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     4501 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     4561 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     4621 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     4681 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     4741 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     4801 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     4861 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     4921 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     4981 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     5041 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     5101 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
     5161 ttgtttaact ttaagaagga gatatacata tgaagtattt cgactgtacg gtcagcgggg
     5221 agcgcggcat catcaagacc tacggaatcc agcttcctga ggaggccctt aaggaacacg
     5281 ttcgtgagta cgttgaaaaa cttcgtgagg gatccgcgat taccatcacc tgcactgccg
     5341 gtgaccgtgt gttcaaattc aaggacaagg ttggctcgtg gggctcgcac caccaccacc
     5401 accactaata ggctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     5461 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatggg
//
