LOCUS       sequence-452101-        4788 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold insertion into the C-terminus of the 
            LAMP1 locus. For lysosome visualization. To be co-transfected with 
            sgRNA plasmid px330-LAMP1 (Addgene #207787).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4788)
  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 4788)
  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-LAMP1
FEATURES             Location/Qualifiers
     source          1..4788
                     /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             1138..1815
                     /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(2567..2583)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2567..2583)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2580..2602)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2591..2607
                     /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(2615..2621,2622..2639,2640..2645))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2660..2681
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2798..2815)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2969..3557)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3049..3068)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3728..4519,4520..4588))
                     /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     4351..4370
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4589..4693)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4761..4779
                     /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 cttggctctg gtgagtgtca
      421 ccgagggcag ctgtcgcggg gtgtggagga cgtgcttcag actccgcctg tggacgttta
      481 gtcgcttccg tgtgggctgg ggcgacgccc ctgttcctct gcaaggagct gtttcttctt
      541 gccggtctga gattctagag gtaactcccc ctgctttaga gaggcccagc gtgtctctca
      601 gctgggagcc cctggtacca tttgagagta agggaatcat tttaagaaac agtggtggcg
      661 cttctcccat gaacgttgaa tacaacactg tcatctgatg tgcacagagg ccctggacgc
      721 agcacagctg tccggccaca gcccctgatt ccagacgggg gagagacgtg tgtggtttcc
      781 tcgccgtgca gagagcacca gtctctgcag cggcttcccc aagtgacaat tccagctaga
      841 gctggaacct tccgccaggg ttcctctttg ccttttggct gatgtggggg agtggtgggg
      901 agagaggcct gtttgcaggc ccctgtgtga gcagagccct gacaccatcc gtctgtcttg
      961 gcagtggagg agtgtctgct ggacgagaac agcatgctga tccccatcgc tgtgggtggt
     1021 gccctggcgg ggctggtcct catcgtcctc atcgcctacc tcgtcggcag gaagaggagt
     1081 cacgcaggct atcagactat cggaggaagc ggaggatctg gcggtaccgg cggaagcatg
     1141 gtgtctacag gcgaggaact gtttaccggc gtggtgccct tcaagttcca gctgaagggc
     1201 accatcaacg gcaagagctt caccgtggaa ggcgagggcg agggcaatag ccacgagggc
     1261 agccacaaag gcaagtatgt gtgcaccagc ggcaaactgc caatgtcttg ggccgccctg
     1321 ggaactagct tcggctatgg catgaaatac tataccaagt accccagcgg cctgaaaaac
     1381 tggttccacg aggtgatgcc tgagggcttc acctacgaca gacacatcca gtacaagggc
     1441 gacggcagca tccacgccaa gcaccagcac ttcatgaaga acggcaccta ccacaacatc
     1501 gtggagttca ccggccagga cttcaaggag aacagccccg tgctgaccgg cgacatggac
     1561 gtgagcctgc ccaacgaggt gcagcacatc cccattgatg acggcgtgga gtgcacagtg
     1621 accctgcagt accctctgct gagcgacgaa agcaagtgcg tggaagccta tcagaacacc
     1681 atcatcaagc ccctgcacaa tcagccagcc cccgatgtgc catttcactg gatcagaaag
     1741 cagtacaccc agagcaagga cgacaccgag gagagagacc acatcatcca gagcgagacc
     1801 ctggaggccc acctgggagg ttccctcgag accggtaaga gcaagtgata gcctggtgca
     1861 cgcatgcaca gcagctgcag gggcctctgt tcctttctct gggcttaggg tcctgtcgaa
     1921 ggggaggcac actttctggc aaacgtttct caaatctgct tcatccaatg tgaagttcat
     1981 cttgcagcat ttactatgca caacagagta actatcgaaa tgacggtgtt aattttgcta
     2041 actgggttaa atattttgct aactggttaa acattaatat ttaccaaagt aggattttga
     2101 gggtgggggt gctctctctg agggggtggg ggtgccgctg tctctgaggg gtgggggtgc
     2161 cgctgtctct gaggggtggg ggtgccgctc tctctgaggg ggtgggggtg ccgctttctc
     2221 tgagggggtg ggggtgccgc tctctctgag ggggtggggg tgctgctctc tccgaggggt
     2281 ggaatgccgc tgtctctgag gggtgggggt gccgctctaa attggctcca tatcatttga
     2341 gtttagggtt ctggtgtttg gtttcttcat tctttactgc actcagattt aagccttaca
     2401 aagggaaagc ctctggccgt cacacgtagg acgcatgaag gtcactcgtg gtgaggctga
     2461 catgctcaca cattacaaca gtagagaggg aaaatcctaa gacagaggaa ctccagagat
     2521 gagtgtctgg agcgcttcag ttcagcttaa gcttggcgta atcatggtca tagctgtttc
     2581 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     2641 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     2701 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     2761 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2821 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2881 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2941 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3001 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3061 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3121 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3181 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3241 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3301 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3361 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3421 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3481 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3541 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3601 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3661 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3721 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3781 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3841 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3901 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3961 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4021 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4081 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4141 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4201 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4261 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4321 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4381 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4441 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4501 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4561 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4621 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4681 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     4741 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
