LOCUS       sequence-479890-        7221 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express RAD21-HaloTag 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 7221)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7221)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: RAD21-HaloTag Vector
FEATURES             Location/Qualifiers
     source          1..7221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    3..84
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        317..620
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        621..824
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     775..795
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          960..1092
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1075..1094
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1136..1155
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1136..1154
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3082..3102
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             3118..4002
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     polyA_signal    4089..4210
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4100..4119)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(4325..4358)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4368..4415
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4447..5241
                     /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(4501..4520)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5111..5130
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      5458..6003
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     5904..5923
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6120..6137
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    6287..6335
                     /note="synthetic polyadenylation signal"
     CDS             complement(6359..6757)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6754..6763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6788..7145)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6802..6937
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6954..7145
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     7102..7122
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 cggcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
       61 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
      121 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct
      181 gattatgatc cgcggccgca atacgtcgac gttatcagct gacttcgtac gagagcctag
      241 gattatggcg cgccactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      301 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      361 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      421 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      481 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      541 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      601 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      661 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      721 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      781 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      841 agatcactag aagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg
      901 cttctgacac aacagtctcg aacttaagct gcagaagttg gtcgtgaggc actgggcagg
      961 taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac
     1021 agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
     1081 ttctctccac aggtgtccac tcccagttca attacagctc ttaaggctag agtattaata
     1141 cgactcacta tagggctagc gatcgccatg ttctacgcac attttgttct cagtaaaaga
     1201 gggcctctgg ccaaaatttg gctagcggcc cattgggata agaagctaac caaagcccat
     1261 gtgttcgagt gtaatttaga gagcagcgtg gagagtatca tctcaccaaa ggtgaaaatg
     1321 gcattacgga catcaggaca tctcttactg ggagtagttc gaatctatca caggaaagcc
     1381 aaataccttc ttgcagactg taatgaagca ttcattaaga taaagatggc ttttcggcca
     1441 ggtgtggttg acctgcctga ggaaaatcgg gaagcagctt ataatgccat tactttacct
     1501 gaagaatttc atgactttga tcagccactg cctgacttag atgacatcga tgtggcccag
     1561 cagttcagct tgaatcagag tagagtggaa gagataacca tgagagaaga agttgggaac
     1621 atcagtattt tacaagaaaa tgattttggt gattttggaa tggatgatcg tgagataatg
     1681 agagaaggca gtgcttttga ggatgacgac atgttagtaa gcactactac ttctaacctc
     1741 ctattagagt ctgaacagag caccagcaat ctgaatgaga aaattaacca tttagaatat
     1801 gaagatcaat ataaggatga taattttgga gaaggaaatg atggtggaat attagatgac
     1861 aaacttatta gtaataatga tggcggtatc tttgatgatc cccctgccct ctctgaggca
     1921 ggggtgatgt tgccagagca gcctgcacat gacgatatgg atgaggatga taatgtatca
     1981 atgggtgggc ctgatagtcc tgattcagtg gatcccgttg aaccaatgcc aaccatgact
     2041 gatcaaacaa cacttgttcc aaatgaggaa gaagcatttg cattggagcc tattgatata
     2101 actgttaaag aaacaaaagc caagaggaag aggaagctaa ttgttgacag tgtcaaagag
     2161 ttggatagca agacaattag agcccaactt agtgattatt cagatattgt tactactttg
     2221 gatctggcac cgcccaccaa gaaattgatg atgtggaaag agacaggagg agtagaaaaa
     2281 ctgttttctt tacctgctca gcctttgtgg aataacagac tactgaagct ctttacacgc
     2341 tgtcttacac cgcttgtacc agaagacctt agaaaaagga ggaaaggagg agaggcagat
     2401 aatttggatg aattcctcaa agaatttgaa aatccagagg ttcctagaga ggaccagcaa
     2461 cagcagcatc agcagcgtga tgttatcgat gagcccatta ttgaagagcc aagccgcctc
     2521 caggagtcag tgatggaggc cagcagaaca aacatagatg agtcagctat gcctccacca
     2581 ccacctcagg gagttaagcg aaaagctgga caaattgacc cagagcctgt gatgcctcct
     2641 cagcaggtag agcagatgga aataccacct gtagagcttc ccccagaaga acctccaaat
     2701 atctgtcagc taataccaga gttagaactt ctgccagaaa aagagaagga gaaagagaag
     2761 gaaaaagaag atgatgaaga ggaagaggat gaagatgcat cagggggcga tcaagatcag
     2821 gaagaaagaa gatggaacaa aaggactcag cagatgcttc atggtcttca gcgtgctctt
     2881 gctaaaactg gagctgaatc tatcagtttg cttgagttat gtcgaaatac gaacagaaaa
     2941 caagctgccg caaagttcta cagcttcttg gttcttaaaa agcagcaagc tattgagctg
     3001 acacaggaag aaccgtacag tgacatcatc gcaacacctg gaccaaggtt ccatattata
     3061 gtttctctcg agccaaccac tgaggatctg tactttcaga gcgataacga tggatccgaa
     3121 atcggtactg gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac
     3181 tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc
     3241 tcctcctacg tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct
     3301 ccagacctga tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac
     3361 cacgtccgct tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc
     3421 attcacgact ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc
     3481 aaaggtattg catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa
     3541 tttgcccgcg agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc
     3601 gatcagaacg tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa
     3661 gtcgagatgg accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg
     3721 cgcttcccaa acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa
     3781 gaatacatgg actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca
     3841 ggcgttctga tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag
     3901 gctgtggaca tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc
     3961 agcgagatcg cgcgctggct gtctactctg gagatttccg gttaatagaa ttctagagtc
     4021 gacctgcagg catgcaagct gatttggctg ctgccaccgc tgagcaaggc cgcttcgagc
     4081 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     4141 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     4201 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg
     4261 ggaggttttt ttaagcaagt aaaacctcta caaatgtggt aaaatcttaa ttaagaattc
     4321 taatataact tcgtatagca tacattatac gaagttatgg atccactgtt gacaattaat
     4381 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa acccaggagg
     4441 cagatcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4501 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4561 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4621 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4681 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4741 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4801 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4861 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4921 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4981 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     5041 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     5101 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5161 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5221 cttcttgacg agttcttctg attcgaaatg accgaccaag cgacgcccaa ccggtatcag
     5281 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5341 tgtcaaacat gagaattaca acttatatcg tatggggctg acttcaggtg ctacatttga
     5401 agagataaat tgcactgaaa tctagaaata ttttatctga ttaataagat gatcttcttg
     5461 agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac gaaaaaaccg ccttgcaggg
     5521 cggtttttcg aaggttctct gagctaccaa ctctttgaac cgaggtaact ggcttggagg
     5581 agcgcagtca ccaaaacttg tcctttcagt ttagccttaa ccggcgcatg acttcaagac
     5641 taactcctct aaatcaatta ccagtggctg ctgccagtgg tgcttttgca tgtctttccg
     5701 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcggactga acggggggtt
     5761 cgtgcataca gtccagcttg gagcgaactg cctacccgga actgagtgtc aggcgtggaa
     5821 tgagacaaac gcggccataa cagcggaatg acaccggtaa accgaaaggc aggaacagga
     5881 gagcgcacga gggagccgcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5941 cgccaccact gatttgagcg tcagatttcg tgatgcttgt caggggggcg gagcctatgg
     6001 aaaaacggct ttgccgcggc cctctcactt ccctgttaag tatcttcctg gcatcttcca
     6061 ggaaatctcc gccccgttcg taagccattt ccgctcgccg cagtcgaacg accgagcgta
     6121 gcgagtcagt gagcgaggaa gcggaatata tcctgtatca catattctgc tgacgcaccg
     6181 gtgcagcctt ttttctcctg ccacatgaag cacttcactg acaccctcat cagtgccaac
     6241 atagtaagcc agtatacact ccgctagcgc tgatgtccgg aatcgtcaca caaaaaacca
     6301 acacacagat gtaatgaaaa taaagatatt ttattgcggc catcgtgatg gctagagttt
     6361 agccctccca cacataacca gagggcagca attcacgaat cccaactgcc gtcggctgtc
     6421 catcactgtc cttcactatg gctttgatcc caggatgcag atcgagaagc acctgtcggc
     6481 accgtccgca ggggctcaag atgcccctgt tctcatttcc gatcgcgacg atacaagtca
     6541 ggttgccagc tgccgcagca gcagcagtgc ccagcaccac gagttctgca caaggtcccc
     6601 cagtaaaatg atatacattg acaccagtga agatgcggcc gtcgctagag agagctgcgc
     6661 tggcgacgct gtagtcttca gagatgggga tgctgttgat tgtagccgtt gctctttcaa
     6721 tgagggtgga ttcttcttga gacaaaggct tggccatggt ggcgctagtg tcagaagaat
     6781 cgagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     6841 cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg
     6901 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
     6961 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     7021 actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     7081 gggagcctgg ggactttcca caccctaact gacacacatt ccacagctgg ttctttccgc
     7141 ctcagaaggt acctaaccaa gttcctcttt cagaggttat ttcaggccat ggtgctgcgc
     7201 aagacgcgtt aattaagacg t
//
