LOCUS       sequence-308004-        7611 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 4E1H.95_LUMABS sensor in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7611)
  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 7611)
  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: 4E1H.95_LUMABS
FEATURES             Location/Qualifiers
     source          1..7611
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..1083
                     /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     complement(20..39)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             1892..2083
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2077..2099)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2185..2324
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2240..2259
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2339..2356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2510..3098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2590..2609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3209..3228)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3220..4035
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             3220..4035
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3293..3312)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4127..4582)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4264..4285)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4476..4495
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4619..4666)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4662..4680
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4733..4750)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4772..4795)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(6950..6961)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(7082..7099)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(7109..7126)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             7145..7167
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    7182..7206
                     /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)."
     primer_bind     complement(7206..7225)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7207..7225)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7309..7328)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7534..7611
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
       61 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
      121 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
      181 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
      241 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
      301 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
      361 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
      421 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
      481 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
      541 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
      601 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
      661 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
      721 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
      781 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
      841 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
      901 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
      961 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     1021 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     1081 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat
     1141 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     1201 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     1261 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     1321 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     1381 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat
     1441 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     1501 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     1561 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     1621 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     1681 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     1741 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     1801 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     1861 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     1921 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     1981 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     2041 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     2101 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     2161 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     2221 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt
     2281 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     2341 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2401 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2461 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2521 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2581 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2641 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2701 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2761 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2821 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2881 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2941 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3001 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3061 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3121 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata
     3181 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     3241 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     3301 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     3361 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     3421 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     3481 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     3541 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     3601 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     3661 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     3721 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     3781 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     3841 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     3901 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     3961 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     4021 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     4081 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     4141 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     4201 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     4261 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     4321 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     4381 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     4441 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     4501 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     4561 aatgcgccgc tacagggcgc gtcccattcg ccaatccgga tatagttcct cctttcagca
     4621 aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat tgctcagcgg
     4681 tggcagcagc caactcagct tcctttcggg ctttgttagc agccggatct cagtggtggt
     4741 ggtggtggtg ctcgagtgcg gccgcctatt atttttcaaa ctgcggatgg ctccagcttc
     4801 ctcccacggg taggggtggc agcggctttg aacgaataga tcctccaccg ctagaagcta
     4861 atattctttc gcacagtcgc caacccgtaa ccccgtttat cgtaactctg aacagaaggc
     4921 tgccatctgg gtttatcaag cgctcatcaa tgattttatt accattccag agcgtcccgg
     4981 taaccgtaat ttttttaccg tcgaacaccg caataccttc ataaggacgc ccaaagtaat
     5041 cgatcatatt cggagtaacg ccatcgataa cgagggtgcc atagtgcagt ataaccttga
     5101 agtgatgatc gtctactggg tacaccactt tgaatatttt ttctatctgc cccatctgat
     5161 caccagaaag accttcgtac gggataatga cgtggatatc gattttcaga ccattctctc
     5221 ccgacagaac gatgcgctga attggcgtga cactcacccc caggttttga aacaggctgc
     5281 taaccccgcc ctgttcaagc acttggtcca ggttatagcc ggcggtctgg cgccaatcac
     5341 cgacaaaatc ttcaagagta aataccatgc tgcctccact agtaccaacc ttatctttga
     5401 acttgaaaac acgatcacca gcagtgcagg tgatggtaat tgccgatcct tcacgcagtt
     5461 tctccacgta ttcgcgaacg tgctctttca gagcctcctc gggaagctgg atgccgtaag
     5521 ttttgatgat tccgcgctca ccagacaccg tgcagtcaaa atacttcccg ccagagccac
     5581 cagatccgcc acttccacct gaaccaccgg aacctccact tcccgcttta gccgctgcct
     5641 ccttggcggc cgcctctttg gctgccgcct cttttgctgc cgcttcctta gcggcagctt
     5701 ccttcgccgc cgcctcagca ccagaacccc cgcttccgcc actccctccg ctgcccccgg
     5761 aaccaccgga tccggccttc gccgcagcct cttttgctgc ggcctctttt gccgcagctt
     5821 ctttcgctgc ggcttcttta gccgcggctt ctttagcggc tgcttctgca ccactacctc
     5881 ctgaaccccc agaacctccc gagccgccag aaccgccaga acccccacct accttatcct
     5941 taaacttgaa tactcgatcc cctgccgtac aagtgattgt tatagcagag ccttccctga
     6001 gtttttcgac gtactcgcga acgtgctcct taagggcctc ttcaggaagt tgtatcccat
     6061 acgtcttgat aatgccgcgc tcgccggaga cggtacagtc aaaatacttg gtacctccgc
     6121 ccgacttata cagttcgtcc attcccatga catccgtaaa tgccttctgc cattccttga
     6181 aattgagctc cgttttagaa tgtttcagtt cggttttacg gaacacatac atcggctggt
     6241 ttttcagata attcgcagcc attggctttg caaacgtgta ggtcgtgcgg gccgttgaac
     6301 gatagcgttt cccgtttcct gttgtatagg accacttaaa agtagaaatg atagttttat
     6361 cgtttggata cgttttcttg gatcggcacc aatcagcagc ggtgaggctg tttgtcatga
     6421 ctgggccatc cgctgggaat ccagttccct tcacctgcgc ttctcccttt atatggctac
     6481 cctcataggt gtacctgtaa tttaccgtca aagacgcccc atcttcaaac tgcatggtcc
     6541 gatgcacttg atatcctgag ccatctacca ttgcggcctg aaaagggctc atgccatccg
     6601 ggtatggaag atactgatgg aaaccataac ctatatgcgg caccaggatc cagggcgaga
     6661 actgcagatc accctttgtg cttttaagat tcaactcctc atatccatcg ttagggttcc
     6721 ccgtaccctg acctaccatg tcgaaatcca ctccgtttat gctcccaaaa atatgaagct
     6781 catgtgtggc aggcagagaa gccatattgt cttcttcacc tttacttacc atatggctgc
     6841 ctgagcctcc cgagccgccc cgatgcattt cctcaggacc gtcaatcaac tgaacataat
     6901 tcgacgggat gatgcctgtc tctccattgg cgcggcgggc tttccaccac cgcccctcaa
     6961 tgtctgatac ctgaaggatt tcattttgtt cgaatgagat ttcgtatgcg tcatcatcat
     7021 ctgcatcata gggatagagc gccttcgcct tgtatataaa gttgtcgtcg ctagccatat
     7081 ggctgccgcg cggcaccagg ccgctgctgt gatgatgatg atgatggctg ctgcccatgg
     7141 tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt atccgctcac
     7201 aattccccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc ctctacgccg
     7261 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
     7321 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
     7381 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac
     7441 cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc
     7501 aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt
     7561 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa t
//
