LOCUS       sequence-263198-        7892 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of N-terminal HaloTag-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 7892)
  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 7892)
  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: pHTNW
FEATURES             Location/Qualifiers
     source          1..7892
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1061..1070
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1067..1957
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1970..1990
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     protein_bind    2081..2205
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2230..2235,2236..2253,2254..2260)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2314..2973
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2380..2399)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3315..3620
                     /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     3499..3518
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3661..3785)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3843..3861)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3893..4014
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3904..3923)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3958..3977
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4259..4616
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4259..4450
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4282..4302)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4467..4602
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4667..5461
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4721..4740)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5331..5350
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5525..5573
                     /note="synthetic polyadenylation signal"
     promoter        5729..5833
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5834..5902,5903..6694)
                     /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(6052..6071)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6852..7440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6932..6951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7556..7839
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcaaa gccaccatgg cagaaatcgg
     1081 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
     1141 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
     1201 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
     1261 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
     1321 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
     1381 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
     1441 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
     1501 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
     1561 gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga
     1621 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     1681 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     1741 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     1801 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     1861 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     1921 gatcgcgcgc tggctgtcga cgctcgagat ttccggcgag ccaaccactg aggatctgta
     1981 ctttcagagc gataacgcga tcgcttccga attcagagct caaccgcgga tatctagatt
     2041 tgggcccaat tcctgcagga ttttgcggcc gcggatctcc acaagtttgt acaaaaaagc
     2101 tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa
     2161 acagactaca taatactgta aaacacaaca tatccagtca ctatggcggc cgcattaggc
     2221 accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggat
     2281 ccgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
     2341 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
     2401 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
     2461 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     2521 catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     2581 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     2641 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     2701 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     2761 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     2821 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     2881 caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
     2941 cagtactgcg atgagtggca gggcggggcg taaacgcgtg gatccggctt actaaaagcc
     3001 agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg
     3061 tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca gtgacagttg
     3121 acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag
     3181 cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca
     3241 ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa
     3301 caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc
     3361 tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc
     3421 tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata
     3481 tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta
     3541 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc
     3601 tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgac
     3661 catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc
     3721 taatttaata tattgatatt tatatcattt tacgtttctc gttcagcttt cttgtacaaa
     3781 gtggtaagta actaagatcc gtcgacctgc agcggccgcg cttgctgagt tggctgctgc
     3841 caccgctgag caataactag cataacccct tggccgcttc gagcagacat gataagatac
     3901 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     3961 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     4021 aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt ttttttaagc
     4081 aagtaaaacc tctacaaatg tggtaaaatc gaattttaac aaaatattaa cgcttacaat
     4141 ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgcgga
     4201 tctgcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct
     4261 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     4321 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     4381 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     4441 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     4501 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     4561 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     4621 ttgattcttc tgacacaaca gtctcgaact taaggctaga gccaccatga ttgaacaaga
     4681 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4741 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4801 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     4861 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4921 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4981 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     5041 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     5101 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     5161 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     5221 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     5281 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     5341 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     5401 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     5461 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgatgg
     5521 ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat
     5581 agcgataagg atcctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac
     5641 attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtaatgg tttcttagac
     5701 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5761 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5821 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5881 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5941 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     6001 gagttttcgc cccgaagaac gttttccaat gatgagcact ttcaaagttc tgctatgtgg
     6061 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     6121 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     6181 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6241 tctgacaact atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6301 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6361 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6421 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6481 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6541 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6601 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6661 tgagataggt gcctcactga ttaagcattg gtaattcgaa atgaccgacc aagcgacgcc
     6721 caaccggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     6781 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     6841 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     6901 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6961 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     7021 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     7081 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     7141 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     7201 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     7261 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     7321 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     7381 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatttcaa
     7441 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     7501 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt atagtccgga
     7561 aatacaggaa cgcacgctgg atggcccttc gctgggatgg tgaaaccatg aaaaatggca
     7621 gcttcagtgg attaagtggg ggtaatgtgg cctgtaccct ctggttgcat aggtattcat
     7681 acggttaaaa tttatcaggc gcgattgcgg cagtttttcg ggtggtttgt tgccattttt
     7741 acctgtctgc tgccgtgatc gcgctgaacg cgttttagcg gtgcgtacaa ttaagggatt
     7801 atggtaaatc cacttactgt ctgccctcgt agccatcgag ataaaccgca gtactccggc
     7861 cacgatgcgt ccggcgtaga ggatcgagat ct
//
