LOCUS       sequence-308006-        7761 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 4E2H.210_LUMABS sensor in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7761)
  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 7761)
  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: 4E2H.210_LUMABS
FEATURES             Location/Qualifiers
     source          1..7761
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(24..71)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     67..85
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(138..155)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(177..200)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(2505..2516)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(2637..2654)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(2664..2681)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2700..2722
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2737..2761
                     /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(2761..2780)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2762..2780)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2864..2883)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3089..3166
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3167..4249
                     /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(3186..3205)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5058..5249
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5243..5265)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5351..5490
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5406..5425
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5505..5522)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5676..6264)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5756..5775)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6375..6394)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6386..7201
                     /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             6386..7201
                     /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(6459..6478)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(7293..7748)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7430..7451)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7642..7661
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg
       61 ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg
      121 ttagcagccg gatctcagtg gtggtggtgg tggtgctcga gtgcggccgc ctattatttt
      181 tcaaactgcg gatggctcca gcttcctccc acgggtaggg gtggcagcgg ctttgaacga
      241 atagatcctc caccgctaga agctaatatt ctttcgcaca gtcgccaacc cgtaaccccg
      301 tttatcgtaa ctctgaacag aaggctgcca tctgggttta tcaagcgctc atcaatgatt
      361 ttattaccat tccagagcgt cccggtaacc gtaatttttt taccgtcgaa caccgcaata
      421 ccttcataag gacgcccaaa gtaatcgatc atattcggag taacgccatc gataacgagg
      481 gtgccatagt gcagtataac cttgaagtga tgatcgtcta ctgggtacac cactttgaat
      541 attttttcta tctgccccat ctgatcacca gaaagacctt cgtacgggat aatgacgtgg
      601 atatcgattt tcagaccatt ctctcccgac agaacgatgc gctgaattgg cgtgacactc
      661 acccccaggt tttgaaacag gctgctaacc ccgccctgtt caagcacttg gtccaggtta
      721 tagccggcgg tctggcgcca atcaccgaca aaatcttcaa gagtaaatac catgctgcct
      781 ccactagtgc tgccttttaa ggaaatggta actgttccgc cctcaacact aacgccctcc
      841 ttcagagcct ttgcaagggc ctgcagcact tcttcgatat tcccatccga cgtcttaatt
      901 attccacgcg aaagtttcgg cacgttggcc gaaaaggtga cttttgaccc gtccagtttc
      961 tttgcccatt ctttccagtg ctcttggacg cattgggcta attcatcgcg cgttggtgca
     1021 gtgcatgaga tagtattcgg gtcgaccccg ccactaccgc cactgccgcc agagccaccg
     1081 gagccgcccg acccaccact tccggctttc gcagccgctt cctttgccgc tgcctctttt
     1141 gccgcggctt ctttagcggc ggcctccttg gccgcagcct ctttggcggc tgcctcagct
     1201 cctgatcccc cgctgccacc tgacccgccg gagccaccac tgccaccaga tccggctttt
     1261 gctgccgcct ctttcgccgc cgcctccttt gcggcagctt ccttcgcagc ggcctcttta
     1321 gctgctgctt ccttggctgc ggcttcagca ccgctccctc cggaaccccc ggagcctcct
     1381 gagccgcctg aaccacccga acccccacgc gtgcttccct tcaggcttat tgtgactgtc
     1441 ccgccttcaa ccgacactcc ctccttcagt gctttagcca gagcttgcag cacctcttcg
     1501 atattgccat ctgaagtttt aatgatgccc cgacttaatt tcgggacgtt cgcagagaac
     1561 gtcactttgc taccgtcaag tttcttcgcc cactccttcc aatgttcttg gacgcactga
     1621 gcaagctcat cacgggtagg ggcggtgcat gagatggtgt tcggggtacc tccgcccgac
     1681 ttatacagtt cgtccattcc catgacatcc gtaaatgcct tctgccattc cttgaaattg
     1741 agctccgttt tagaatgttt cagttcggtt ttacggaaca catacatcgg ctggtttttc
     1801 agataattcg cagccattgg ctttgcaaac gtgtaggtcg tgcgggccgt tgaacgatag
     1861 cgtttcccgt ttcctgttgt ataggaccac ttaaaagtag aaatgatagt tttatcgttt
     1921 ggatacgttt tcttggatcg gcaccaatca gcagcggtga ggctgtttgt catgactggg
     1981 ccatccgctg ggaatccagt tcccttcacc tgcgcttctc cctttatatg gctaccctca
     2041 taggtgtacc tgtaatttac cgtcaaagac gccccatctt caaactgcat ggtccgatgc
     2101 acttgatatc ctgagccatc taccattgcg gcctgaaaag ggctcatgcc atccgggtat
     2161 ggaagatact gatggaaacc ataacctata tgcggcacca ggatccaggg cgagaactgc
     2221 agatcaccct ttgtgctttt aagattcaac tcctcatatc catcgttagg gttccccgta
     2281 ccctgaccta ccatgtcgaa atccactccg tttatgctcc caaaaatatg aagctcatgt
     2341 gtggcaggca gagaagccat attgtcttct tcacctttac ttaccatatg gctgcctgag
     2401 cctcccgagc cgccccgatg catttcctca ggaccgtcaa tcaactgaac ataattcgac
     2461 gggatgatgc ctgtctctcc attggcgcgg cgggctttcc accaccgccc ctcaatgtct
     2521 gatacctgaa ggatttcatt ttgttcgaat gagatttcgt atgcgtcatc atcatctgca
     2581 tcatagggat agagcgcctt cgccttgtat ataaagttgt cgtcgctagc catatggctg
     2641 ccgcgcggca ccaggccgct gctgtgatga tgatgatgat ggctgctgcc catggtatat
     2701 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
     2761 ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc
     2821 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
     2881 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
     2941 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc
     3001 cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag
     3061 tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg
     3121 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     3181 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     3241 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     3301 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     3361 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     3421 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     3481 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     3541 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     3601 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     3661 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     3721 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     3781 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     3841 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     3901 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     3961 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     4021 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     4081 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     4141 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4201 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4261 ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct
     4321 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     4381 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg
     4441 gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa
     4501 tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga
     4561 agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt
     4621 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac
     4681 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa
     4741 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca
     4801 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg
     4861 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata
     4921 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc
     4981 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc
     5041 cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta
     5101 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg
     5161 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc
     5221 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     5281 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     5341 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     5401 ctatgcggca tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac
     5461 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     5521 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5581 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5641 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5701 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5761 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5821 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5881 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5941 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6001 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6061 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     6121 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6181 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6241 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6301 cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta
     6361 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag
     6421 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     6481 tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt
     6541 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     6601 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     6661 tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga
     6721 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     6781 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     6841 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     6901 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     6961 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     7021 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     7081 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     7141 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     7201 agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7261 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     7321 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     7381 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     7441 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     7501 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     7561 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     7621 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     7681 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     7741 gggcgcgtcc cattcgccaa t
//
