LOCUS       sequence-488903-        6460 bp    DNA     circular SYN 09-OCT-2025
DEFINITION  Express ER-HaloTag(R) Fusion Protein in Mammalian Cells under a CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6460)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6460)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 9, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: NanoBRET Assay Acceptor Vector, ER
FEATURES             Location/Qualifiers
     source          1..6460
                     /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"
     CDS             1911..2846
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             1959..2849
                     /codon_start=1
                     /gene="ESR1"
                     /product="estrogen nuclear receptor alpha ligand-binding 
                     domain"
                     /label=ER-LBD
                     /translation="KRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM
                     GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKL
                     LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF
                     LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNK
                     GMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSL
                     QKYYITGEAEGFPATV"
     CDS             2871..2891
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             2907..3791
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(3879..3897)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3934..4055
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3945..3964)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3999..4018
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             4454..5248
                     /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="MLEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4508..4527)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5118..5137
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5420..6008)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5500..5519)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6124..6407
                     /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 ggctagcgat cgccatgacc atgaccctcc
     1081 acaccaaagc atccgggatg gccctactgc atcagatcca agggaacgag ctggagcccc
     1141 tgaaccgtcc gcagctcaag atccccctgg agcggcccct gggcgaggtg tacctggaca
     1201 gcagcaagcc cgccgtgtac aactaccccg agggcgccgc ctacgagttc aacgccgcgg
     1261 ccgccgccaa cgcgcaggtc tacggtcaga ccggcctccc ctacggcccc gggtctgagg
     1321 ctgcggcgtt cggctccaac ggcctggggg gtttcccccc actcaacagc gtgtctccga
     1381 gcccgctgat gctactgcac ccgccgccgc agctgtcgcc tttcctgcag ccccacggcc
     1441 agcaggtgcc ctactacctg gagaacgagc ccagcggcta cacggtgcgc gaggccggcc
     1501 cgccggcatt ctacaggcca aattcagata atcgacgcca gggtggcaga gaaagattgg
     1561 ccagtaccaa tgacaaggga agtatggcta tggaatctgc caaggagact cgctactgtg
     1621 cagtgtgcaa tgactatgct tcaggctacc attatggagt ctggtcctgt gagggctgca
     1681 aggccttctt caagagaagt attcaaggac ataacgacta tatgtgtcca gccaccaacc
     1741 agtgcaccat tgataaaaac aggaggaaga gctgccaggc ctgccggctc cgcaaatgct
     1801 acgaagtggg aatgatgaaa ggtgggatac gaaaagaccg aagaggaggg agaatgttga
     1861 aacacaagcg ccagagagat gatggggagg gcaggggtga agtggggtct gctggagaca
     1921 tgagagctgc caacctttgg ccaagcccgc tcatgatcaa acgctctaag aagaacagcc
     1981 tggccttgtc cctgacggcc gaccagatgg tcagtgcctt gttggatgct gagcccccga
     2041 tactctattc cgagtatgat cctaccagac ccttcagtga agcttcgatg atgggcttac
     2101 tgaccaacct ggcagacagg gagctggttc acatgatcaa ctgggcgaag agggtgccag
     2161 gctttgtgga tttgaccctc catgatcagg tccaccttct agaatgtgcc tggctagaga
     2221 tcctgatgat tggtctcgtc tggcgctcca tggagcaccc agggaagcta ctgtttgctc
     2281 ctaacttgct cttggacagg aaccagggaa aatgtgtaga gggcatggtg gagatcttcg
     2341 acatgctgct ggctacatca tctcggttcc gcatgatgaa tctgcaggga gaggagtttg
     2401 tgtgcctcaa atctattatt ttgcttaatt ctggagtgta cacatttctg tccagcaccc
     2461 tgaagtctct ggaagagaag gaccatatcc accgagtcct ggacaagatc acagacactt
     2521 tgatccacct gatggccaag gcaggcctga ccctgcagca gcagcaccag cggctggccc
     2581 agctcctcct catcctctcc cacatcaggc acatgagtaa caaaggcatg gagcatctgt
     2641 acagcatgaa gtgcaagaac gtggtgcccc tctatgacct gctgctggag atgctggacg
     2701 cccaccgcct acatgcgccc actagccgtg gaggggcatc cgtggaggag acggaccaaa
     2761 gccacttggc cactgcgggc tctacttcat cgcattcctt gcaaaagtat tacatcacgg
     2821 gggaggcaga gggtttccct gccacggtcg tttctctcga gccaaccact gaggatctgt
     2881 actttcagag cgataacgat ggatccgaaa tcggtactgg ctttccattc gacccccatt
     2941 atgtggaagt cctgggcgag cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc
     3001 ctgtgctgtt cctgcacggt aacccgacct cctcctacgt gtggcgcaac atcatcccgc
     3061 atgttgcacc gacccatcgc tgcattgctc cagacctgat cggtatgggc aaatccgaca
     3121 aaccagacct gggttatttc ttcgacgacc acgtccgctt catggatgcc ttcatcgaag
     3181 ccctgggtct ggaagaggtc gtcctggtca ttcacgactg gggctccgct ctgggtttcc
     3241 actgggccaa gcgcaatcca gagcgcgtca aaggtattgc atttatggag ttcatccgcc
     3301 ctatcccgac ctgggacgaa tggccagaat ttgcccgcga gaccttccag gccttccgca
     3361 ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc
     3421 cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga ccattaccgc gagccgttcc
     3481 tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa cgagctgcca atcgccggtg
     3541 agccagcgaa catcgtcgcg ctggtcgaag aatacatgga ctggctgcac cagtcccctg
     3601 tcccgaagct gctgttctgg ggcaccccag gcgttctgat cccaccggcc gaagccgctc
     3661 gcctggccaa aagcctgcct aactgcaagg ctgtggacat cggcccgggt ctgaatctgc
     3721 tgcaagaaga caacccggac ctgatcggca gcgagatcgc gcgctggctg tctactctgg
     3781 agatttccgg ttaatagaat tctagagtcg acctgcaggc atgcaagctg atccggctgc
     3841 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     3901 accccttggg gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa
     3961 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     4021 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     4081 tgtttcaggt tcagggggag atgtgggagg tttttttaag caagtaaaac ctctacaaat
     4141 gtggtaaaat cgaattctaa tggatcctct ttgcgcttgc gttttccctt gtccagatag
     4201 cccagtagct gacattcatc cggggtcagc accgtttctg cggactggct ttctacgtgt
     4261 tccgcttcct ttagcagccc ttgcgccctg agtgcttgcg gcagcgtgag cttcaaaaga
     4321 attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc
     4381 tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag agacaggatg
     4441 acggtcgttt cgcatgcttg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4501 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4561 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4621 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4681 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4741 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4801 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4861 agcgaaacat cgcatcgagc gagcacgcac tcggatggaa gccggtcttg tcgatcagga
     4921 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4981 gcgtatgccg gatggtgagg atctcgtcgt gactcatggc gatgcctgct tgccgaatat
     5041 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     5101 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     5161 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     5221 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     5281 gcgacgccca accggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5341 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5401 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5461 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5521 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5581 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5641 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5701 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5761 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5821 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     5881 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5941 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6001 gatttcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6061 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttat
     6121 agtccggaaa tacaggaacg cacgctggat ggcccttcgc tgggatggtg aaaccatgaa
     6181 aaatggcagc ttcagtggat taagtggggg taatgtggcc tgtaccctct ggttgcatag
     6241 gtattcatac ggttaaaatt tatcaggcgc gattgcggca gtttttcggg tggtttgttg
     6301 ccatttttac ctgtctgctg ccgtgatcgc gctgaacgcg ttttagcggt gcgtacaatt
     6361 aagggattat ggtaaatcca cttactgtct gccctcgtag ccatcgagat aaaccgcagt
     6421 actccggcca cgatgcgtcc ggcgtagagg atcgagatct
//
