LOCUS       sequence-417172-        7164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterially expressed HiBiT-FLuc for Ni-NTA purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7164)
  AUTHORS   Ciulla DA, Dranchak PK, Aitha M, van Neer RHP, Shah D, Tharakan R, 
            Wilson KM, Wang Y, Braisted JC, Inglese J
  TITLE     A general assay platform to study protein pharmacology using 
            ligand-dependent structural dynamics.
  JOURNAL   Nat Commun. 2025 May 10;16(1):4342. doi: 10.1038/s41467-025-59658-6.
  PUBMED    40346061
REFERENCE   2  (bases 1 to 7164)
  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"; journalName: "Nat 
            Commun."; date: "2025-05-10"; volume: "16(1):4342. doi"; pages: " 
            10.1038/s41467-025-59658-6"
COMMENT     Sequence Label: FLuc-N-HiBiT
FEATURES             Location/Qualifiers
     source          1..7164
                     /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(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(167..196)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             complement(206..223)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(230..1876)
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
                     IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
                     NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
                     YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
                     DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
                     QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
                     LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
                     YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
                     QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
                     VDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     1795..1813
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             complement(1928..1960)
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     RBS             1968..1990
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2005..2029
                     /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(2029..2048)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2030..2048)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2132..2151)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2357..2434
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2435..3517
                     /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(2454..2473)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4326..4517
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4511..4533)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4619..4761
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4674..4693
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4776..4793)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4947..5535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5027..5046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5706..6497,6498..6566))
                     /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     6329..6348
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6567..6671)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6698..7153)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6835..6856)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7047..7066
                     /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 ggtggtgctc gagtcaatga tgatgatggt
      181 ggtggtggtg gtggtgccct gcagcgcttc cgcgaggcac taaaagcttc acggcgatct
      241 ttccgccctt cttggccttt atgaggatct ctctgatttt tcttgcgtcg agttttccgg
      301 taagaccttt cggtacttcg tccacaaaca caactcctcc gcgcaacttt ttcgcggttg
      361 ttacttgact ggcgacgtaa tccacgatct ctttttccgt catcgtcttt ccgtgctcca
      421 aaacaacaac ggcggcggga agttcaccgg cgtcatcgtc gggaagacct gcgacacctg
      481 cgtcgaagat gttggggtgt tggagcaaga tggattccaa ttcagcggga gccacctgat
      541 agcctttgta cttaatcaga gacttcaggc ggtcaacgat gaagaagtgt tcgtcttcgt
      601 cccagtaagc tatgtctcca gaatgtagcc atccatcctt gtcaatcaag gcgttggtcg
      661 cttccggatt gtttacataa ccggacataa tcataggacc tctcacacac agttcgcctc
      721 tttgattaac gcccagcgtt ttcccggtat ccagatccac aaccttcgct tcaaaaaatg
      781 gaacaacttt accgaccgcg cccggtttat catccccctc gggtgtaatc agaatagctg
      841 atgtagtctc agtgagccca tatccttgcc tgatacctgg cagatggaac ctcttggcaa
      901 ccgcttcccc gacttcctta gagaggggag cgccaccaga agcaatttcg tgtaaattag
      961 ataaatcgta tttgtcaatc agagtgcttt tggcgaagaa ggagaatagg gttggcacca
     1021 gcagcgcact ttgaatcttg taatcctgaa ggctcctcag aaacagctct tcttcaaatc
     1081 tatacattaa gacgactcga aatccacata tcaaatatcc gagtgtagta aacattccaa
     1141 aaccgtgatg gaatggaaca acacttaaaa tcgcagtatc cggaatgatt tgattgccaa
     1201 aaataggatc tctggcatgc gagaatctca cgcaggcagt tctatgaggc agagcgacac
     1261 ctttaggcag accagtagat ccagaggagt tcatgatcag tgcaattgtc ttgtccctat
     1321 cgaaggactc tggcacaaaa tcgtattcat taaaaccggg aggtagatga gatgtgacga
     1381 acgtgtacat cgactgaaat ccctggtaat ccgttttaga atccatgata ataatttttt
     1441 ggatgattgg gagctttttt tgcacgttca aaattttttg caaccccttt ttggaaacga
     1501 acaccacggt aggctgcgaa atgcccatac tgttgagcaa ttcacgttca ttataaatgt
     1561 cgttcgcggg cgcaactgca actccgataa ataacgcgcc caacaccggc ataaagaatt
     1621 gaagagagtt ttcactgcat acgacgattc tgtgatttgt attcagccca tatcgtttca
     1681 tagcttctgc caaccgaacg gacatttcga agtactcagc gtaagtgatg tccacctcga
     1741 tatgtgcatc tgtaaaagca attgttccag gaaccagggc gtatctcttc atagccttat
     1801 gcagttgctc tccagcggtt ccatcttcca gcggatagaa tggcgccggg cctttcttta
     1861 tgtttttggc gtcttcgagc tcgctaatct tcttgaacag ccgccagccg ctcaccatgg
     1921 atccgcgacc catttgctgt ccaccagtca tgctagccat atgtatatct ccttcttaaa
     1981 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
     2041 tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc
     2101 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
     2161 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
     2221 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
     2281 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     2341 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     2401 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     2461 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     2521 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     2581 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     2641 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     2701 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     2761 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     2821 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     2881 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     2941 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     3001 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     3061 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     3121 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     3181 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     3241 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     3301 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     3361 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     3421 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3481 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     3541 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
     3601 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     3661 tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct
     3721 ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc
     3781 tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta
     3841 tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc
     3901 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
     3961 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
     4021 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
     4081 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
     4141 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
     4201 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
     4261 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
     4321 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
     4381 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
     4441 aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg
     4501 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4561 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     4621 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     4681 agagcagatt gtactgagag tgcaccatat atgcggtgtg aaataccgca cagatgcgta
     4741 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4801 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4861 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4921 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4981 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5041 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5101 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5161 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5221 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5281 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5341 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     5401 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5461 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5521 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5581 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5641 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5701 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5761 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5821 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5881 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5941 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6001 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6061 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6121 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6181 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6241 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6301 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6361 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6421 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6481 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6541 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6601 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6661 ggggttccgc gcacatttcc ccgaaaagtg ccacctgaaa ttgtaaacgt taatattttg
     6721 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     6781 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     6841 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     6901 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     6961 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     7021 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     7081 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     7141 ctacagggcg cgtcccattc gcca
//
