LOCUS       sequence-416383-        5628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for moxGFP-2A-Puro 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 5628)
  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 5628)
  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-moxGFP-Puro-LAMP1
FEATURES             Location/Qualifiers
     source          1..5628
                     /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             join(1129..1131,1132..1134,1135..1845)
                     /codon_start=1
                     /product="monomeric GFP variant optimized for use in 
                     oxidizing environments such as the secretory pathway 
                     (Costantini et al., 2015)"
                     /label=moxGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1174..1195)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1782..1803
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1855..1881
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1855..1881
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1885..1911
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1918..1951)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1963..2019
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2026..2622
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2026..2045)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2522..2542
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2629..2662)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3407..3423)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3407..3423)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3420..3442)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3431..3447
                     /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(3455..3461,3462..3479,3480..3485))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3500..3521
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3638..3655)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3809..4397)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3889..3908)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4568..5359,5360..5428))
                     /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     5191..5210
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5429..5533)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5601..5619
                     /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 gcggtaccat ggtgagcaag
     1141 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtgaac
     1201 ggccacaagt tctccgtgcg gggcgagggc gagggcgatg ccaccaacgg caagctgacc
     1261 ctgaagttca tcagcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1321 ctgacctacg gcgtgcagag cttctcccgc taccccgacc acatgaagcg ccacgacttc
     1381 ttcaagagcg ccatgcccga aggctacgtc caggagcgca ccatctcctt caaggacgac
     1441 ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1501 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1561 aacttcaact cccacaacgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     1621 aacttcaaga tccgccataa cgtggaggac ggctccgtgc agctcgccga ccactaccag
     1681 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgtccacc
     1741 cagtccaagc tgtccaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1801 gtgaccgccg ccgggatcac tcacggcatg gacgagctgt acaagggctc tggctacccc
     1861 tacgacgtgc ccgactacgc cggctatccg tatgatgtcc cggactatgc aacgcgttac
     1921 cgttcgtata gcatacatta tacgaagtta tctggatccg gagctactaa cttcagcctg
     1981 ctgaagcagg ccggagacgt ggaggagaac cctggacctt ctagaatgac cgagtacaag
     2041 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     2101 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     2161 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     2221 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     2281 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     2341 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     2401 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     2461 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     2521 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     2581 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cccccgggat aacttcgtat
     2641 agcatacatt atacgaacgg tagcctcgag accggtaaga gcaagtgata gcctggtgca
     2701 cgcatgcaca gcagctgcag gggcctctgt tcctttctct gggcttaggg tcctgtcgaa
     2761 ggggaggcac actttctggc aaacgtttct caaatctgct tcatccaatg tgaagttcat
     2821 cttgcagcat ttactatgca caacagagta actatcgaaa tgacggtgtt aattttgcta
     2881 actgggttaa atattttgct aactggttaa acattaatat ttaccaaagt aggattttga
     2941 gggtgggggt gctctctctg agggggtggg ggtgccgctg tctctgaggg gtgggggtgc
     3001 cgctgtctct gaggggtggg ggtgccgctc tctctgaggg ggtgggggtg ccgctttctc
     3061 tgagggggtg ggggtgccgc tctctctgag ggggtggggg tgctgctctc tccgaggggt
     3121 ggaatgccgc tgtctctgag gggtgggggt gccgctctaa attggctcca tatcatttga
     3181 gtttagggtt ctggtgtttg gtttcttcat tctttactgc actcagattt aagccttaca
     3241 aagggaaagc ctctggccgt cacacgtagg acgcatgaag gtcactcgtg gtgaggctga
     3301 catgctcaca cattacaaca gtagagaggg aaaatcctaa gacagaggaa ctccagagat
     3361 gagtgtctgg agcgcttcag ttcagcttaa gcttggcgta atcatggtca tagctgtttc
     3421 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3481 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3541 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3601 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3661 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3721 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3781 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3841 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3901 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3961 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4021 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4081 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4141 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4201 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4261 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4321 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4381 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4441 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4501 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4561 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4621 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4681 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4741 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4801 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4861 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4921 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4981 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5041 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5101 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5161 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5221 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5281 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5341 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5401 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5461 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5521 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     5581 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
