LOCUS       sequence-451303-        4488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold insertion into the C-terminus of the 
            EZR locus. For membrane visualization. To be co-transfected with 
            sgRNA plasmid px330-EZR (Addgene #227293).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4488)
  AUTHORS   Philip R, Sharma A, Matellan L, Erpf AC, Hsu WH, Tkach JM, Wyatt 
            HDM, Pelletier L
  TITLE     qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous 
            tagging.
  JOURNAL   EMBO J. 2024 Dec 12. doi: 10.1038/s44318-024-00337-5.
  PUBMED    39668248
REFERENCE   2  (bases 1 to 4488)
  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: "EMBO J. 
            2024 Dec 12. doi: 10.1038/s44318-024-00337-5."
COMMENT     Sequence Label: qTAG-C-Solo-mStayGold-EZR
FEATURES             Location/Qualifiers
     source          1..4488
                     /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"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             988..1665
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ando et al., 2023)"
                     /label=mStayGold(J)
                     /note="includes multiple mutations plus an N-terminal 
                     iinker sequence"
                     /translation="MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     primer_bind     complement(2267..2283)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2267..2283)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2280..2302)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2291..2307
                     /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(2315..2321,2322..2339,2340..2345))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2360..2381
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2498..2515)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2669..3257)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2749..2768)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3428..4219,4220..4288))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4051..4070
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4289..4393)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4461..4479
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     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 ccagtgaatt cacctggtga tgacagcacc
      421 cccgccccca ccaccccccg tgtacgagcc ggtgagctac catgtccagg agagcttgca
      481 ggatgagggc gcagagccca cgggctacag cgcggagctg tctagtgagg gcatccggga
      541 tgaccgcaat gaggagaagc gcatcactga ggcagagaag aacgagcgtg tgcagcggca
      601 gctgctggtg aggcccacgg ggctctgtct ccagggagcc tgcctcgctt ttggagcaga
      661 caggcttggg gagggcagtg atgtgagcca gccccaccca gcacccctct tgcccttcct
      721 gttttcctag gggacgggcc gggccatatg gggaggaagg gactagacca acgctgctta
      781 atgttacaga cgctgagcag cgagctgtcc caggcccgag atgagaataa gaggacccac
      841 aatgacatca tccacaacga gaacatgagg caaggccggg acaagtacaa gacgctgcgg
      901 cagatccggc agggcaacac caagcagcgc atcgacgagt tcgaggccct gggaggaagc
      961 ggaggatctg gcggtaccgg cggaagcatg gtgtctacag gcgaggaact gtttaccggc
     1021 gtggtgccct tcaagttcca gctgaagggc accatcaacg gcaagagctt caccgtggaa
     1081 ggcgagggcg agggcaatag ccacgagggc agccacaaag gcaagtatgt gtgcaccagc
     1141 ggcaaactgc caatgtcttg ggccgccctg ggaactagct tcggctatgg catgaaatac
     1201 tataccaagt accccagcgg cctgaaaaac tggttccacg aggtgatgcc tgagggcttc
     1261 acctacgaca gacacatcca gtacaagggc gacggcagca tccacgccaa gcaccagcac
     1321 ttcatgaaga acggcaccta ccacaacatc gtggagttca ccggccagga cttcaaggag
     1381 aacagccccg tgctgaccgg cgacatggac gtgagcctgc ccaacgaggt gcagcacatc
     1441 cccattgatg acggcgtgga gtgcacagtg accctgcagt accctctgct gagcgacgaa
     1501 agcaagtgcg tggaagccta tcagaacacc atcatcaagc ccctgcacaa tcagccagcc
     1561 cccgatgtgc catttcactg gatcagaaag cagtacaccc agagcaagga cgacaccgag
     1621 gagagagacc acatcatcca gagcgagacc ctggaggccc acctgggagg ttccctcgag
     1681 accggtaaga gcaagtgata acagccatgc aaggaccaag ggcagagggg tgctcatagc
     1741 gggcgctgcc agccccgcca cgcttgtgtc tttagtgctc caagtctagg aactccctca
     1801 gatcccagtt cctttagaaa gcagttaccc aacagaaaca ttctgggctg ggaaccaggg
     1861 aggcgccctg gtttgttttc cccagttgta atagtgccaa gcaggcctga ttctcgcgat
     1921 tattctcgaa tcacctcctg tgttgtgctg ggagcaggac tgattgaatt acggaaaatg
     1981 cctgtaaagt ctgagtaaga aacttcatgc tggcctgtgt gatacaagag tcagcatcat
     2041 taaaggaaac gtggcaggac ttccatctgt gccatacttg ttctgtattc gaaatgagct
     2101 caaattgatt ttttaatttc tatgaaggat ccatctttgt atatttacat gcttagaggg
     2161 gtgaaaatta ttttggaaat tgagtctgaa gcactctcgc acacacagtg attccctcct
     2221 cccgtcactc cacgcagctg gcagagagaa gcttggcgta atcatggtca tagctgtttc
     2281 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     2341 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     2401 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2461 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2521 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2581 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2641 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2701 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2761 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2821 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2881 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2941 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3001 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3061 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3121 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3181 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3241 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3301 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3361 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3421 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3481 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3541 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3601 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3661 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3721 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3781 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3841 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3901 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3961 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4021 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4081 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4141 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4201 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4261 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4321 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4381 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     4441 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
