LOCUS       sequence-272878-        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        119..498
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        499..702
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     653..673
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          838..970
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     953..972
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1014..1033
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1014..1032
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1042..1051
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1048..1938
                     /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             1951..1971
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     protein_bind    2062..2186
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2211..2216,2217..2234,2235..2241)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2295..2954
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2361..2380)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3296..3601
                     /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     3480..3499
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3642..3766)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3824..3842)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3874..3995
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3885..3904)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3939..3958
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4240..4597
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4240..4431
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4263..4283)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4448..4583
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4648..5442
                     /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(4702..4721)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5312..5331
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5506..5554
                     /note="synthetic polyadenylation signal"
     promoter        5710..5814
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5815..5883,5884..6675)
                     /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(6033..6052)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6833..7421)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6913..6932)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7537..7820
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 atattattca ttggttatat agcataaatc aatattggct attggccatt gcatacgttg
       61 tatctatatc ataatatgta catttatatt ggctcatgtc caatatgacc gccatgttgg
      121 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      181 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      241 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      301 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      361 gtgtatcata tgccaagtcc gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      421 cattatgccc agtacatgac cttacgggac tttcctactt ggcagtacat ctacgtatta
      481 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca ccaatgggcg tggatagcgg
      541 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      601 caccaaaatc aacgggactt tccaaaatgt cgtaataacc ccgccccgtt gacgcaaatg
      661 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
      721 atcactagaa gctttattgc ggtagtttat cacagttaaa ttgctaacgc agtcagtgct
      781 tctgacacaa cagtctcgaa cttaagctgc agaagttggt cgtgaggcac tgggcaggta
      841 agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag
      901 agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt
      961 ctctccacag gtgtccactc ccagttcaat tacagctctt aaggctagag tattaatacg
     1021 actcactata gggctagcaa agccaccatg gcagaaatcg gtactggctt tccattcgac
     1081 ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat
     1141 ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc
     1201 atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg tatgggcaaa
     1261 tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc
     1321 atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg
     1381 ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc
     1441 atccgcccta tcccgacctg ggacgaatgg ccagaatttg cccgcgagac cttccaggcc
     1501 ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt tatcgagggt
     1561 acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg agatggacca ttaccgcgag
     1621 ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc
     1681 gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag
     1741 tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa
     1801 gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg
     1861 aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctgtcg
     1921 acgctcgaga tttccggcga gccaaccact gaggatctgt actttcagag cgataacgcg
     1981 atcgcttccg aattcagagc tcaaccgcgg atatctagat ttgggcccaa ttcctgcagg
     2041 attttgcggc cgcggatctc cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat
     2101 gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt
     2161 aaaacacaac atatccagtc actatggcgg ccgcattagg caccccaggc tttacacttt
     2221 atgcttccgg ctcgtataat gtgtggattt tgagttagga tccgtcgaga ttttcaggag
     2281 ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat
     2341 ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga
     2401 ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt
     2461 atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg
     2521 caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc
     2581 atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt
     2641 ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta
     2701 aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt
     2761 ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat
     2821 attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt
     2881 gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc
     2941 agggcggggc gtaaacgcgt ggatccggct tactaaaagc cagataacag tatgcgtatt
     3001 tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa
     3061 aaagaggtat gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt
     3121 gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat gcagaatgaa
     3181 gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat ggctgaggtc
     3241 gcccggttta ttgaaatgaa cggctctttt gctgacgaga acaggggctg gtgaaatgca
     3301 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
     3361 tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
     3421 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
     3481 catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga
     3541 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
     3601 aatgtcaggc tcccttatac acagccagtc tgcaggtcga ccatagtgac tggatatgtt
     3661 gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat
     3721 ttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggtaagt aactaagatc
     3781 cgtcgacctg cagcggccgc gcttgctgag ttggctgctg ccaccgctga gcaataacta
     3841 gcataacccc ttggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa
     3901 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     3961 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     4021 tgtttcaggt tcagggggag atgtgggagg tttttttaag caagtaaaac ctctacaaat
     4081 gtggtaaaat cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc
     4141 tccttacgca tctgtgcggt atttcacacc gcatacgcgg atctgcgcag caccatggcc
     4201 tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa agaaccagct
     4261 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4321 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     4381 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     4441 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4501 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4561 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag cttgattctt ctgacacaac
     4621 agtctcgaac ttaaggctag agccaccatg attgaacaag atggattgca cgcaggttct
     4681 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4741 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4801 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4861 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4921 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4981 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     5041 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     5101 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     5161 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     5221 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     5281 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     5341 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     5401 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     5461 aaatgaccga ccaagcgacg cccaacctgc catcacgatg gccgcaataa aatatcttta
     5521 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagcgataag gatcctcttt
     5581 gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg gggtcagcac
     5641 cgtttctgcg gactggcttt ctacgtaatg gtttcttaga cgtcaggtgg cacttttcgg
     5701 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5761 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5821 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5881 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5941 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6001 cgttttccaa tgatgagcac tttcaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6061 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6121 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6181 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac tatcggagga
     6241 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6301 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6361 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6421 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6481 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6541 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6601 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6661 attaagcatt ggtaattcga aatgaccgac caagcgacgc ccaaccggta tcagctcact
     6721 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6781 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6841 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6901 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6961 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     7021 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     7081 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     7141 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     7201 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     7261 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     7321 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     7381 tttgcaagca gcagattacg cgcagaaaaa aaggatttca agaagatcct ttgatctttt
     7441 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     7501 tatcaaaaag gatcttcacc tagatccttt tatagtccgg aaatacagga acgcacgctg
     7561 gatggccctt cgctgggatg gtgaaaccat gaaaaatggc agcttcagtg gattaagtgg
     7621 gggtaatgtg gcctgtaccc tctggttgca taggtattca tacggttaaa atttatcagg
     7681 cgcgattgcg gcagtttttc gggtggtttg ttgccatttt tacctgtctg ctgccgtgat
     7741 cgcgctgaac gcgttttagc ggtgcgtaca attaagggat tatggtaaat ccacttactg
     7801 tctgccctcg tagccatcga gataaaccgc agtactccgg ccacgatgcg tccggcgtag
     7861 aggatcgaga tcttcaatat tggccattag cc
//
