LOCUS       sequence-431537-        2740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (C' terminus) Nucleoplasmin NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2740)
  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 2740)
  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: AA009
FEATURES             Location/Qualifiers
     source          1..2740
                     /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..654)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHAGLCSRRARDPMKTCFRHSRGHHMGMHNQARRRDKR
                     NLEHRMGRIPFSFLPGRPFSWPPAFWTRLHLDVFARYRARILWPSFYNVVTGKTLALPN
                     LIALQHIPLSPAGVIAKRPAPIALPNSCAA"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             444..491
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             498..578
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(650..666)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(650..666)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(663..685)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    674..690
                     /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(698..704,705..722,723..728))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    743..764
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(881..898)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1052..1640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1132..1151)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1818..2627)
                     /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     2541..2560
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2619..2638
                     /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 tccaaaaggc cggcggccac gaaaaaggcc ggccaggcaa
      481 aaaagaaaaa gggatcctac ccatacgatg ttccagatta cgcttatccc tacgacgtgc
      541 ctgattatgc atacccatat gatgtccccg actatgccgg aagcacgtct tcatcggatc
      601 ccgggcccgt cgactgcaga ggcctgcatg caagcttggt gtaatcatgg tcatagctgt
      661 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
      721 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
      781 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
      841 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
      901 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
      961 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1021 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1081 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     1141 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1201 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     1261 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     1321 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     1381 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     1441 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     1501 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     1561 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     1621 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     1681 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     1741 agatcctttt aaattaaaaa tgaagtttta aatcaagccc aatctgaata atgttacaac
     1801 caattaacca attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc
     1861 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     1921 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     1981 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     2041 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     2101 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     2161 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     2221 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     2281 tcacctgaat caggatattc ttctaatacc tggaatgctg tttttccggg gatcgcagtg
     2341 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     2401 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     2461 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaagcg atagattgtc
     2521 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     2581 ttggaattta atcgcggcct cgacgtttcc cgttgaatat ggctcataac accccttgta
     2641 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     2701 atgtaacatc agagattttg agacacgggc cagagctgca
//
