LOCUS       sequence-431544-        3532 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (C' terminus) SV40NLS; Linker; MeCP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3532)
  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 3532)
  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: AA134
FEATURES             Location/Qualifiers
     source          1..3532
                     /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"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             444..464
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             513..1370
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
                     /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
                     GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
                     TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
                     HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
                     KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
                     TERVS"
     primer_bind     complement(1442..1458)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1442..1458)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1455..1477)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1466..1482
                     /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(1490..1496,1497..1514,1515..1520))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1535..1556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1673..1690)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1844..2432)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1924..1943)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2610..3419)
                     /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     3333..3352
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3411..3430
                     /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 tccccaaaga aaaagcggaa agtaagcgga agtgagaccc
      481 caggtacatc cgaatcagca acgcctgaaa gcgtgcaggt gaaaagggtg ctggaaaaat
      541 cccccggcaa actcctcgtg aagatgccct tccaggcttc ccctggcgga aaaggtgaag
      601 ggggtggcgc aaccacatct gcccaggtca tggtcatcaa gcgacctgga aggaaaagaa
      661 aggccgaggc tgaccctcag gccattccaa agaaacgggg acgcaagcca gggtccgtgg
      721 tcgcagctgc agcagctgag gctaagaaaa aggcagtgaa ggaaagctcc atccgcagtg
      781 tgcaggagac tgtcctgccc atcaagaaga gaaagactag ggagaccgtg tccatcgagg
      841 tcaaagaagt ggtcaagccc ctgctcgtgt ccaccctggg cgaaaaatct ggaaaggggc
      901 tcaaaacatg caagtcacct ggacggaaaa gcaaggagtc tagtccaaag gggcgctcaa
      961 gctccgcttc tagtccccct aaaaaggaac accatcacca tcaccatcac gccgagtctc
     1021 ctaaggctcc tatgccactg ctcccaccac ctccaccacc tgagccacag tcaagcgagg
     1081 accccatcag cccacccgag cctcaggatc tgtcctctag tatttgcaaa gaggaaaaga
     1141 tgcccagagc aggcagcctg gagagtgatg gctgtccaaa agaacccgcc aagacccagc
     1201 ctatggtggc agccgctgca actaccacca caaccacaac taccacagtg gccgaaaaat
     1261 acaagcatcg cggcgagggc gaacgaaagg acattgtgtc aagctccatg cccagaccta
     1321 accgggagga accagtcgat agtaggacac ccgtgactga gagagtctca ggaagcacgt
     1381 cttcatcgga tcccgggccc gtcgactgca gaggcctgca tgcaagcttg gtgtaatcat
     1441 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     1501 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     1561 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     1621 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     1681 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     1741 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     1801 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1861 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1921 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1981 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2041 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2101 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2161 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2221 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2281 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2341 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2401 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2461 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2521 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaagc ccaatctgaa
     2581 taatgttaca accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac
     2641 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     2701 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     2761 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     2821 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     2881 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     2941 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     3001 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     3061 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgtttttccg
     3121 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     3181 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     3241 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaag
     3301 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3361 tcagcatcca tgttggaatt taatcgcggc ctcgacgttt cccgttgaat atggctcata
     3421 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     3481 ttatcttgtg caatgtaaca tcagagattt tgagacacgg gccagagctg ca
//
