LOCUS       sequence-360258-        5043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for PUF9R-mRuby2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5043)
  AUTHORS   Clow PA, Du M, Jillette N, Taghbalout A, Zhu JJ, Cheng AW
  TITLE     CRISPR-mediated multiplexed live cell imaging of nonrepetitive 
            genomic loci with one guide RNA per locus.
  JOURNAL   Nat Commun. 2022 Apr 6;13(1):1871. doi: 10.1038/s41467-022-29343-z.
  PUBMED    35387989
REFERENCE   2  (bases 1 to 5043)
  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: "2022-04-6"; volume: "13(1):1871. doi"; pages: " 
            10.1038/s41467-022-29343-z"
COMMENT     Sequence Label: pAC11003-pmax-PUF9R_mRuby2
FEATURES             Location/Qualifiers
     source          1..5043
                     /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          813..945
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     protein_bind    960..984
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1002..1011
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1023..1043
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2250..2267
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2271..2303
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(2307..2315,2316..2330)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             2337..3047
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     protein_bind    complement(3072..3096)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3149..3190
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3216..3235)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3245..3326
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3270..3289
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3524..4112)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3604..3623)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4193..4987)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(4193..4987)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4304..4323)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4914..4933
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
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 taagcagagg
      721 tcgtttagtg aaccgtcaga tcactagtag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctc gactgatcac aggtaagtat caaggttaca agacaggttt
      841 aaggaggcca atagaaactg ggcttgtcga gacagagaag attcttgcgt ttctgatagg
      901 cacctattgg tcttactgac atccactttg cctttctctc cacagggaaa aaacaattga
      961 caagtttgta caaaaaagca ggctccgaat tcaccggtgc cgccaccatg gggatcctcc
     1021 cccccaagaa aaagaggaag gtatctagag gccgcagccg ccttttggaa gattttcgaa
     1081 acaaccggta ccccaattta caactgcggg agattgctgg acatataatg gaattttccc
     1141 aagaccagca tgggtccaga ttcattcagc tgaaactgga gcgtgccaca ccagctgagc
     1201 gccagcttgt cttcaatgaa atcctccagg ctgcctacca actcatggtg gatgtgtttg
     1261 gtaattacgt cattcagaag ttctttgaat ttggcagtct tgaacagaag ctggctttgg
     1321 cagaacggat tcgaggccac gtcctgtcat tggcactaca gatgtatggc tgccgtgtta
     1381 tccagaaagc tcttgagttt attccttcag accagcagaa tgagatggtt cgggaactcc
     1441 accaacacac ggagcaactg gtccaggatc aatacggcaa ctacgtgatt cagcacgtgt
     1501 tggagcacgg taggccagag gacaagagta aaattgtcgc cgagatccgg ggcaatgttc
     1561 tggtcctctc tcagcacaaa ttcgcctcca atgttgtgga aaagtgtgtg acccacgcat
     1621 ccagaactga acgagctgtg ctcatcgacg aggtttgtac gatgaatgac ggtcctcact
     1681 ctgcacttta tacaatgatg aaggaccagt acgccaatta tgttgtgcag aaaatgatcg
     1741 acgtagcaga acccggacag cgcaaaatcg tgatgcacga gcttcaccag cacacagagc
     1801 agctggtaca ggatcaatat ggaaattatg taatccaaca tgtactggag cacggtcgtc
     1861 ctgaggataa aagcaaaatt gtagcagaaa tccgaggcaa tgtacttgta ttgagtcagc
     1921 acaaatttgc aagcaatgtt gtggagaagt gtgttactca cgcctcacgt acggagcgcg
     1981 ctgtgctcat cgacgaggtg tgcaccatga acgacggtcc ccacagtgcc ttatacacca
     2041 tgatgaagga ccagtatgcc aactacgtgg tccagaagat gattgacgtg gcggagccag
     2101 gccagcggaa gatcgtcatg cataagatcc ggccccacat cgcaactctt cgtaagtaca
     2161 cctatggcaa gcacattctg gccaagctgg agaagtacta catgaagaac ggtgttgact
     2221 taggggggcc ggccatggtg cggggttctc atcatcatca tcatcatggt atggctagca
     2281 tgactggtgg acagcaaatg ggtcgggatc tgtacgacga tgacgataag gatccgatgg
     2341 tgtctaaggg cgaagagctg atcaaggaaa atatgcgtat gaaggtggtc atggaaggtt
     2401 cggtcaacgg ccaccaattc aaatgcacag gtgaaggaga aggcaatccg tacatgggaa
     2461 ctcaaaccat gaggatcaaa gtcatcgagg gaggacccct gccatttgcc tttgacattc
     2521 ttgccacgtc gttcatgtat ggcagccgta cttttatcaa gtacccgaaa ggcattcctg
     2581 atttctttaa acagtccttt cctgagggtt ttacttggga aagagttacg agatacgaag
     2641 atggtggagt cgtcaccgtc atgcaggaca ccagccttga ggatggctgt ctcgtttacc
     2701 acgtccaagt cagaggggta aactttccct ccaatggtcc cgtgatgcag aagaagacca
     2761 agggttggga gcctaataca gagatgatgt atccagcaga tggtggtctg aggggataca
     2821 ctcatatggc actgaaagtt gatggtggtg gccatctgtc ttgctctttc gtaacaactt
     2881 acaggtcaaa aaagaccgtc gggaacatca agatgcccgg tatccatgcc gttgatcacc
     2941 gcctggaaag gttagaggaa agtgacaatg aaatgttcgt agtacaacgc gaacacgcag
     3001 ttgccaagtt cgccgggctt ggtggtggga tggacgagct gtacaagatc gattgattaa
     3061 ttaagaattc gacccagctt tcttgtacaa agtggttgat atccagcaca gtggcggccg
     3121 ctcgagtcta gagggcccgc ggttcgaagg taagcctatc cctaaccctc tcctcggtct
     3181 cgattctacg cgtaccggtt agcaattgtt ttttcgatga gtttggacaa accacaacta
     3241 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     3301 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     3361 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtactt
     3421 aagaggggga gaccaaaggg cgagacgtta aggcctcacg tgacatgtga gcaaaaggcc
     3481 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3541 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3601 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3661 tgccgcttac gggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3721 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3781 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3841 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3901 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3961 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4021 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4081 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4141 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgccg tctcagaaga
     4201 actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa
     4261 gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca
     4321 acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
     4381 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
     4441 cctcgccgtc gggcatgctc gccttgagcc tggcgaacag ttcggctggc gcgagcccct
     4501 gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc
     4561 tctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca
     4621 gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
     4681 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
     4741 cgtcgagtac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct
     4801 cgtcttgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
     4861 cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
     4921 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
     4981 caatcataat attattgaag catttatcag ggttcgtctc gtcccggtct cctcccatgc
     5041 atg
//
