LOCUS       sequence-387320-        5648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the de novo luciferase, LuxSit-i.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5648)
  AUTHORS   Yeh AH, Norn C, Kipnis Y, Tischer D, Pellock SJ, Evans D, Ma P, Lee 
            GR, Zhang JZ, Anishchenko I, Coventry B, Cao L, Dauparas J, Halabiya
            S, DeWitt M, Carter L, Houk KN, Baker D
  TITLE     De novo design of luciferases using deep learning.
  JOURNAL   Nature. 2023 Feb;614(7949):774-780. doi: 10.1038/s41586-023-05696-3.
            Epub 2023 Feb 22.
  PUBMED    36813896
REFERENCE   2  (bases 1 to 5648)
  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/s41586-023-05696-3"; journalName: "Nature"; date: 
            "2023-02"; volume: "614"; issue: "7949"; pages: "774-780"
COMMENT     Sequence Label: pET29_LuxSit_i
FEATURES             Location/Qualifiers
     source          1..5648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(518..538)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(551..568)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             585..607
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    622..646
                     /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(646..665)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(647..665)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(753..772)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        978..1055
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1056..2138
                     /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(1075..1094)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2947..3138
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3132..3154)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3240..3379
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3295..3314
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3394..3411)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3565..4153)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3645..3664)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4264..4283)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4275..5090
                     /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             4275..5090
                     /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(4348..4367)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5182..5637)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5319..5340)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5531..5550
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtgctcgag ttagccggtc ggattgatat
      181 gcacacgaac ttcggtcaca cgattgccac gaaaatgcca atgatgggtc aaatctacgg
      241 tatgtttctg gccattatgg gtcgcgtgca actgcacatg cacttccacg gtatcgccac
      301 gtacttccag gctcttaatt tcacgctgcg catctttact ggtgctaaac agacgttcaa
      361 accattcacg aaattcttca cggctggtaa aggtaacgcc atcccacaga tgaattgtca
      421 cgcccggatg aaacaggctc gcagcggtat ccgcatcgcc gctatccagc gcttcataaa
      481 aacgacgcag aaactgacga atctgttctt cgctcattcc ttggaagtac aagttctcgc
      541 taccgctacc atggtgatgg tggtgatggc tacccatatg tatatctcct tcttaaagtt
      601 aaacaaaatt atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg
      661 tattaatttc gcgggatcga gatcgatctc gatcctctac gccggacgca tcgtggccgg
      721 catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga
      781 agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg
      841 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc
      901 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg
      961 agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     1021 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     1081 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     1141 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     1201 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     1261 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     1321 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     1381 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     1441 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     1501 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     1561 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     1621 taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     1681 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     1741 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     1801 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggacatct
     1861 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     1921 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     1981 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     2041 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     2101 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     2161 tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc
     2221 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     2281 atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc
     2341 tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc
     2401 ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt
     2461 atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg
     2521 ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg
     2581 tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt
     2641 ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg
     2701 atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag
     2761 cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt
     2821 ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc
     2881 atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag
     2941 gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag
     3001 acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt
     3061 gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat
     3121 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     3181 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     3241 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
     3301 cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag
     3361 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3421 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3481 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3541 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3601 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3661 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3721 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3781 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3841 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3901 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3961 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     4021 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4081 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4141 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4201 aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta
     4261 atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa
     4321 attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat
     4381 caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac
     4441 atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga
     4501 cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt
     4561 tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa tatcctgatt
     4621 caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg
     4681 tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa
     4741 tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg
     4801 aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc
     4861 atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg
     4921 atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc
     4981 tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc
     5041 ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc
     5101 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5161 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     5221 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     5281 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     5341 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     5401 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     5461 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     5521 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     5581 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     5641 attcgcca
//
