LOCUS       sequence-271260-        7668 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of N-terminal NanoLuc luciferase fused proteins in 
            mammalian cells. Genes can be cloned using Gateway cloning system. 
            Cloned vectors can be used for NanoBRET assay..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7668)
  AUTHORS   Yang JS, Garriga-Canut M, Link N, Carolis C, Broadbent K, 
            Beltran-Sastre V, Serrano L, Maurer SP
  TITLE     rec-YnH enables simultaneous many-by-many detection of direct 
            protein-protein and protein-RNA interactions.
  JOURNAL   Nat Commun. 2018 Sep 14;9(1):3747. doi: 10.1038/s41467-018-06128-x.
  PUBMED    30217970
REFERENCE   2  (bases 1 to 7668)
  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: "2018-09-14"; volume: "9(1):3747. doi"; pages: " 
            10.1038/s41467-018-06128-x"
COMMENT     Sequence Label: pNLF1W
FEATURES             Location/Qualifiers
     source          1..7668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        114..471
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        114..305
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(137..157)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      322..457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             502..1539
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             1518..1526
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    1563..1611
                     /note="synthetic polyadenylation signal"
     promoter        1767..1871
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1872..1940,1941..2732)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2090..2109)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(2890..3478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2970..2989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    3594..3877
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     enhancer        4068..4447
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4448..4651
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4602..4622
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          4787..4919
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4902..4921
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     4963..4982
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4963..4981
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4995..5507
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     protein_bind    5604..5728
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(5753..5758,5759..5776,5777..5783)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             5837..6496
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5903..5922)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             6838..7143
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     7022..7041
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(7184..7308)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7366..7384)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    7416..7537
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7427..7446)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7481..7500
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gcggatctgc
       61 gcagcaccat ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc
      121 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
      181 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
      241 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
      301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      361 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcgat
      481 tcttctgaca ctagcgccac catgaagaag cccgaactca ccgctaccag cgttgaaaaa
      541 tttctcatcg agaagttcga cagtgtgagc gacctgatgc agttgtcgga gggcgaagag
      601 agccgagcct tcagcttcga tgtcggcgga cgcggctatg tactgcgggt gaatagctgc
      661 gctgatggct tctacaaaga ccgctacgtg taccgccact tcgccagcgc tgcactaccc
      721 atccccgaag tgttggacat cggcgagttc agcgagagcc tgacatactg catcagtaga
      781 cgcgcccaag gcgttactct ccaagacctc cccgaaacag agctgcctgc tgtgttacag
      841 cctgtcgccg aagctatgga tgctattgcc gccgccgacc tcagtcaaac cagcggcttc
      901 ggcccattcg ggccccaagg catcggccag tacacaacct ggcgggattt catttgcgcc
      961 attgctgatc cccatgtcta ccactggcag accgtgatgg acgacaccgt gtccgccagc
     1021 gtagctcaag ccctggacga actgatgctg tgggccgaag actgtcccga ggtgcgccac
     1081 ctcgtccatg ccgacttcgg cagcaacaac gtcctgaccg acaacggccg catcaccgcc
     1141 gtaatcgact ggtccgaagc tatgttcggg gacagtcagt acgaggtggc caacatcttc
     1201 ttctggcggc cctggctggc ttgcatggag cagcagactc gctacttcga gcgccggcat
     1261 cccgagctgg ccggcagccc tcgtctgcga gcctacatgc tgcgcatcgg cctggatcag
     1321 ctctaccaga gcctcgtgga cggcaacttc gacgatgctg cctgggctca aggccgctgc
     1381 gatgccatcg tccgcagcgg ggccggcacc gtcggtcgca cacaaatcgc tcgccggagc
     1441 gcagccgtat ggaccgacgg ctgcgtcgag gtgctggccg acagcggcaa ccgccggccc
     1501 agtacacgac cgcgcgctaa ggaggtaggt cgagtttaga ctctagccat cacgatggcc
     1561 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
     1621 cgataaggat cctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat
     1681 tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtaatggtt tcttagacgt
     1741 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1801 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1861 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1921 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1981 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     2041 gttttcgccc cgaagaacgt tttccaatga tgagcacttt caaagttctg ctatgtggcg
     2101 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     2161 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     2221 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     2281 tgacaactat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     2341 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     2401 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     2461 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     2521 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     2581 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     2641 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     2701 agataggtgc ctcactgatt aagcattggt aattcgaaat gaccgaccaa gcgacgccca
     2761 accggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2821 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2881 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2941 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3001 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3061 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3121 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3181 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3241 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3301 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     3361 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaaaaaa ccaccgctgg
     3421 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatttcaaga
     3481 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3541 gattttggtc atgagattat caaaaaggat cttcacctag atccttttat agtccggaaa
     3601 tacaggaacg cacgctggat ggcccttcgc tgggatggtg aaaccatgaa aaatggcagc
     3661 ttcagtggat taagtggggg taatgtggcc tgtaccctct ggttgcatag gtattcatac
     3721 ggttaaaatt tatcaggcgc gattgcggca gtttttcggg tggtttgttg ccatttttac
     3781 ctgtctgctg ccgtgatcgc gctgaacgcg ttttagcggt gcgtacaatt aagggattat
     3841 ggtaaatcca cttactgtct gccctcgtag ccatcgagat aaaccgcagt actccggcca
     3901 cgatgcgtcc ggcgtagagg atcgagatct tcaatattgg ccattagcca tattattcat
     3961 tggttatata gcataaatca atattggcta ttggccattg catacgttgt atctatatca
     4021 taatatgtac atttatattg gctcatgtcc aatatgaccg ccatgttggc attgattatt
     4081 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     4141 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     4201 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     4261 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     4321 gccaagtccg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     4381 gtacatgacc ttacgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     4441 taccatggtg atgcggtttt ggcagtacac caatgggcgt ggatagcggt ttgactcacg
     4501 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     4561 acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg
     4621 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tcactagaag
     4681 ctttattgcg gtagtttatc acagttaaat tgctaacgca gtcagtgctt ctgacacaac
     4741 agtctcgaac ttaagctgca gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt
     4801 tacaagacag gtttaaggag accaatagaa actgggcttg tcgagacaga gaagactctt
     4861 gcgtttctga taggcaccta ttggtcttac tgacatccac tttgcctttc tctccacagg
     4921 tgtccactcc cagttcaatt acagctctta aggctagagt attaatacga ctcactatag
     4981 ggctagcgct caccatggtc ttcacactcg aagatttcgt tggggactgg cgacagacag
     5041 ccggctacaa cctggaccaa gtccttgaac agggaggtgt gtccagtttg tttcagaatc
     5101 tcggggtgtc cgtaactccg atccaaagga ttgtcctgag cggtgaaaat gggctgaaga
     5161 tcgacatcca tgtcatcatc ccgtatgaag gtctgagcgg cgaccaaatg ggccagatcg
     5221 aaaaaatttt taaggtggtg taccctgtgg atgatcatca ctttaaggtg atcctgcact
     5281 atggcacact ggtaatcgac ggggttacgc cgaacatgat cgactatttc ggacggccgt
     5341 atgaaggcat cgccgtgttc gacggcaaaa agatcactgt aacagggacc ctgtggaacg
     5401 gcaacaaaat tatcgacgag cgcctgatca accccgacgg ctccctgctg ttccgagtaa
     5461 ccatcaacgg agtgaccggc tggcggctgt gcgaacgcat tctggcgggc tcgagcggcg
     5521 cgatcgcttc cgaattcaga gctcaaccgc ggatatctag atttgggccc aattcctgca
     5581 ggattttgcg gccgcggatc tccacaagtt tgtacaaaaa agctgaacga gaaacgtaaa
     5641 atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact
     5701 gtaaaacaca acatatccag tcactatggc ggccgcatta ggcaccccag gctttacact
     5761 ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatccgtcga gattttcagg
     5821 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca
     5881 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca
     5941 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt
     6001 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat
     6061 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt
     6121 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca
     6181 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc
     6241 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag
     6301 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa
     6361 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
     6421 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg
     6481 gcagggcggg gcgtaaacgc gtggatccgg cttactaaaa gccagataac agtatgcgta
     6541 tttgcgcgct gatttttgcg gtataagaat atatactgat atgtataccc gaagtatgtc
     6601 aaaaagaggt atgctatgaa gcagcgtatt acagtgacag ttgacagcga cagctatcag
     6661 ttgctcaagg catatatgat gtcaatatct ccggtctggt aagcacaacc atgcagaatg
     6721 aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg atggctgagg
     6781 tcgcccggtt tattgaaatg aacggctctt ttgctgacga gaacaggggc tggtgaaatg
     6841 cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag
     6901 agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg
     6961 ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg
     7021 cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct
     7081 gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata
     7141 taaatgtcag gctcccttat acacagccag tctgcaggtc gaccatagtg actggatatg
     7201 ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat
     7261 atttatatca ttttacgttt ctcgttcagc tttcttgtac aaagtggtaa gtaactaaga
     7321 tccgtcgacc tgcagcggcc gcgcttgctg agttggctgc tgccaccgct gagcaataac
     7381 tagcataacc ccttggccgc ttcgagcaga catgataaga tacattgatg agtttggaca
     7441 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     7501 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     7561 tatgtttcag gttcaggggg agatgtggga ggttttttta agcaagtaaa acctctacaa
     7621 atgtggtaaa atcgaatttt aacaaaatat taacgcttac aatttcct
//
