LOCUS       sequence-367380-        4860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for Clover-PUF48107.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4860)
  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 4860)
  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: pAC11004-pmax-Clover_PUF48107
FEATURES             Location/Qualifiers
     source          1..4860
                     /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             join(1008..1010,1011..1013,1014..1724)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1053..1074)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1661..1682
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1740..1758)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1741..1758)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1797..1817
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(2889..2913)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2966..3007
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3033..3052)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3062..3143
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3087..3106
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3341..3929)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3421..3440)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4010..4804)
                     /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(4010..4804)
                     /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(4121..4140)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4731..4750
                     /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 gtgagcaagg
     1021 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1081 gccacaagtt cagcgtccgc ggcgagggcg agggcgatgc caccaacggc aagctgaccc
     1141 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct
     1201 tcggctacgg cgtggcctgc ttcagccgct accccgacca catgaagcag cacgacttct
     1261 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctctttc aaggacgacg
     1321 gtacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1381 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1441 acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaagaacggc atcaaggcta
     1501 acttcaagat ccgccacaac gttgaggacg gcagcgtgca gctcgccgac cactaccagc
     1561 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagccatc
     1621 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1681 tgaccgccgc cgggattaca catggcatgg acgagctgta caagtctaga gggccctatt
     1741 ctatagtgtc acctaaatgc ggaggcggcg gtagcgggcc ggccgggatc ctccccccca
     1801 agaaaaagag gaaggtatct agaggccgca gccgcctttt ggaagatttt cgaaacaacc
     1861 ggtaccccaa tttacaactg cgggagattg ctggacatat aatggaattt tcccaagacc
     1921 agcatgggag cagattcatt gagctgaaac tggagcgtgc cacaccagct gagcgccagc
     1981 ttgtcttcaa tgaaatcctc caggctgcct accaactcat ggtggatgtg tttggtaatt
     2041 acgtcattca gaagttcttt gaatttggca gtcttgaaca gaagctggct ttggcagaac
     2101 ggattcgagg ccacgtcctg tcattggcac tacagatgta tggcagccgt gttatcgaga
     2161 aagctcttga gtttattcct tcagaccagc agaatgagat ggttcgggaa ctagatggcc
     2221 atgtcttgaa gtgtgtgaaa gatcagaatg gcaatcacgt ggttcagaaa tgcattgaat
     2281 gtgtacagcc ccagtctttg caatttatca tcgacgcgtt taagggacag gtatttgcct
     2341 tatccacaca tccttatggc aacagagtga ttcagagaat cctggagcac tgtctccctg
     2401 accagacact ccctatttta gaggagcttc accagcacac agagcagctg gtacaggatc
     2461 aatatggaag ctacgtaatc gagcatgtac tggagcacgg tcgtcctgag gataaaagca
     2521 aaattgtagc agaaatccga ggcaatgtac ttgtattgag tcagcacaaa tttgcaagca
     2581 atgttgtgga gaagtgtgtt actcacgcct cacgtacgga gcgcgctgtg ctcatcgacg
     2641 aggtgtgcac catgaacgac ggtccccaca gtgccttata caccatgatg aaggaccagt
     2701 atgccaacta cgtggtccag aagatgattg acgtggcgga gccaggccag cggaagatcg
     2761 tcatgcataa gatccggccc cacatcgcaa ctcttcgtaa gtacacctat ggcaagcaca
     2821 ttctggccaa gctggagaag tactacatga agaacggtgt tgacttaggg tgattaatta
     2881 agaattcgac ccagctttct tgtacaaagt ggttgatatc cagcacagtg gcggccgctc
     2941 gagtctagag ggcccgcggt tcgaaggtaa gcctatccct aaccctctcc tcggtctcga
     3001 ttctacgcgt accggttagc aattgttttt tcgatgagtt tggacaaacc acaactagaa
     3061 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     3121 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     3181 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtacttaag
     3241 agggggagac caaagggcga gacgttaagg cctcacgtga catgtgagca aaaggccagc
     3301 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3361 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3421 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3481 cgcttacggg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3541 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3601 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3661 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3721 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3781 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3841 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3901 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3961 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgccgtct cagaagaact
     4021 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     4081 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     4141 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     4201 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
     4261 cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     4321 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctctct
     4381 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     4441 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     4501 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     4561 cgagtacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     4621 cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     4681 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     4741 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     4801 tcataatatt attgaagcat ttatcagggt tcgtctcgtc ccggtctcct cccatgcatg
//
