LOCUS       sequence-431467-        3064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (C' terminus) ALFA; ALFA; ALFA; ALFA; ALFA; NpNLS;
            3xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3064)
  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 3064)
  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: AA197
FEATURES             Location/Qualifiers
     source          1..3064
                     /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             468..506
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             531..569
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             594..632
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             657..695
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             720..758
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             768..815
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             822..902
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(974..990)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(974..990)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(987..1009)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    998..1014
                     /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(1022..1028,1029..1046,1047..1052))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1067..1088
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1205..1222)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1376..1964)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1456..1475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2142..2951)
                     /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     2865..2884
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2943..2962
                     /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 tccggagggc cgagctctgg cgcacccagc agactggagg
      481 aggagctgag aagaaggctg accgagccac caagtggagg gtctcctgcc tccagactcg
      541 aggaagagct cagaaggaga ctcaccgaag ggtccccaac atctactgaa gaatctaggc
      601 tcgaagagga actcaggagg aggctcacag aaggcggacc ttcctcagga gccccttcac
      661 ggctggaaga agaactgcgg aggcgcctca ctgagccatc ttccacaggg ggaccagcca
      721 gtcgcctcga agaagagctc aggcggagac tgacagaggg caccagcaaa aggccggcgg
      781 ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc ctacccatac gatgttccag
      841 attacgctta tccctacgac gtgcctgatt atgcataccc atatgatgtc cccgactatg
      901 ccggaagcac gtcttcatcg gatcccgggc ccgtcgactg cagaggcctg catgcaagct
      961 tggtgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     1021 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     1081 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1141 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     1201 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     1261 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1321 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1381 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     1441 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1501 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     1561 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1621 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1681 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1741 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1801 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1861 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1921 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     1981 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     2041 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     2101 gcccaatctg aataatgtta caaccaatta accaattctg attagaaaaa ctcatcgagc
     2161 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     2221 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2281 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2341 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2401 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2461 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2521 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2581 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2641 gctgtttttc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2701 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2761 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2821 ttcccataca agcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2881 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgacgt ttcccgttga
     2941 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3001 gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac gggccagagc
     3061 tgca
//
