LOCUS       sequence-436825-        2839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (C' terminus) VP64; NpNLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2839)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 2839)
  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: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA019
FEATURES             Location/Qualifiers
     source          1..2839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(250..753)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHAGLCSRRARDPMKTCFPFSFLPGRPFSWPPAFWSLR
                     SACPGRNHQGRHCRACRGQSHQGHPNQACQGQNHRGRHFPAYPDQNRPMRRSLRSRRSR
                     TRLHLDVFARYRARILWPSFYNVVTGKTLALPNLIALQHIPLSPAGVIAKRPAPIALPN
                     SCAA"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             468..617
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             630..677
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(749..765)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(749..765)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(762..784)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    773..789
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(797..803,804..821,822..827))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    842..863
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(980..997)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1151..1739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1231..1250)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1917..2726)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2640..2659
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2718..2737
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagacggg tccgggagcg gcgggagcgg agggagcgac gcattggacg
      481 attttgatct ggatatgctg ggaagtgacg ccctcgatga ttttgacctt gacatgcttg
      541 gttcggatgc ccttgatgac tttgacctcg acatgctcgg cagtgacgcc cttgatgatt
      601 tcgacctgga catgctgagc ggaggctcca aaaggccggc ggccacgaaa aaggccggcc
      661 aggcaaaaaa gaaaaaggga agcacgtctt catcggatcc cgggcccgtc gactgcagag
      721 gcctgcatgc aagcttggtg taatcatggt catagctgtt tcctgtgtga aattgttatc
      781 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
      841 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
      901 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
      961 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1021 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     1081 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     1141 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     1201 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     1261 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     1321 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     1381 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     1441 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     1501 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     1561 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     1621 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     1681 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     1741 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     1801 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     1861 gaagttttaa atcaagccca atctgaataa tgttacaacc aattaaccaa ttctgattag
     1921 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     1981 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     2041 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     2101 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     2161 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     2221 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     2281 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     2341 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     2401 tctaatacct ggaatgctgt ttttccgggg atcgcagtgg tgagtaacca tgcatcatca
     2461 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     2521 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     2581 tctggcgcat cgggcttccc atacaagcga tagattgtcg cacctgattg cccgacatta
     2641 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc
     2701 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     2761 agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga
     2821 gacacgggcc agagctgca
//
