LOCUS       sequence-451695-        5308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold-2A-Puro insertion into the C-terminus 
            of the CLTC locus. For clathrin heavy chain visualization. To be 
            co-transfected with sgRNA px330-PITCh-CLTC (Addgene #227312).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5308)
  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 5308)
  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-mStayGold-Puro-CLTC
FEATURES             Location/Qualifiers
     source          1..5308
                     /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             992..1669
                     /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"
     CDS             1685..1726
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1748..1781)
                     /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             1793..1849
                     /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             1856..2452
                     /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(1856..1875)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2352..2372
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2459..2492)
                     /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(3087..3103)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3087..3103)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3100..3122)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3111..3127
                     /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(3135..3141,3142..3159,3160..3165))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3180..3201
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3318..3335)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3489..4077)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3569..3588)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4248..5039,5040..5108))
                     /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     4871..4890
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5109..5213)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5281..5299
                     /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 cgaggtttca gtgagccgag
      421 attacacaac tgcactccag cctaggcaac agagccagat tctttttttt tttttttttt
      481 aaaaagtctt ttaaaaaatt cttttattgt gctgatttta ttgtgtcatg aagtgtaata
      541 tgcatgtagg tatgtgtcaa gtatacagag tgtcaggcat acggtgttca gtcataagca
      601 gttctggcct ttggccctgc actgtttgtt ggctttttag gtaggaacct tcttagagta
      661 agactgtcat gctaaaattg tagcaatcaa atgtgccccc catacaactc atttgaggtt
      721 gagattatgt tgctagagtg gaggagattg gagtgttcta aatgctaaca gtttgtcctg
      781 cctccttgcc cctgccccct gctccttttt gttttcttct actgtaggtc agccccagtt
      841 gatgctgaca gcaggaccca gtgttgccgt ccctccccag gcaccttttg gttatggtta
      901 taccgcacca ccgtatggac agccacagcc cggattcgga tattctatgg gaggaagcgg
      961 aggatctggc ggtaccggag gtggcggaag catggtgtct acaggcgagg aactgtttac
     1021 cggcgtggtg cccttcaagt tccagctgaa gggcaccatc aacggcaaga gcttcaccgt
     1081 ggaaggcgag ggcgagggca atagccacga gggcagccac aaaggcaagt atgtgtgcac
     1141 cagcggcaaa ctgccaatgt cttgggccgc cctgggaact agcttcggct atggcatgaa
     1201 atactatacc aagtacccca gcggcctgaa aaactggttc cacgaggtga tgcctgaggg
     1261 cttcacctac gacagacaca tccagtacaa gggcgacggc agcatccacg ccaagcacca
     1321 gcacttcatg aagaacggca cctaccacaa catcgtggag ttcaccggcc aggacttcaa
     1381 ggagaacagc cccgtgctga ccggcgacat ggacgtgagc ctgcccaacg aggtgcagca
     1441 catccccatt gatgacggcg tggagtgcac agtgaccctg cagtaccctc tgctgagcga
     1501 cgaaagcaag tgcgtggaag cctatcagaa caccatcatc aagcccctgc acaatcagcc
     1561 agcccccgat gtgccatttc actggatcag aaagcagtac acccagagca aggacgacac
     1621 cgaggagaga gaccacatca tccagagcga gaccctggag gcccacctgg gaggtggcgg
     1681 ttccggcaag cccatcccca accccctgct gggcctggac agcaccggag gtggcggatc
     1741 aacgcgttac cgttcgtata gcatacatta tacgaagtta tctggatccg gagctactaa
     1801 cttcagcctg ctgaagcagg ccggagacgt ggaggagaac cctggacctt ctagaatgac
     1861 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     1921 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     1981 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     2041 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     2101 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     2161 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     2221 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     2281 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     2341 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     2401 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cccccgggat
     2461 aacttcgtat agcatacatt atacgaacgg tagcctcgag accggtaaga gcaagtgatg
     2521 agatgaagcg ctgatcctgt agtcacctat tttcgtactg aaacatcgtc tttacccact
     2581 tctcagttta taatggggga aaacaggcaa cgtgttcttg taacctttat ttcatgaagg
     2641 acttcttttt gtttctaact ataaacttgg atcacctatg ttaaaacctt atttcacatt
     2701 ccacatcatt ttagaattta ttttcgaagg ggaatagttt caatgtttta ttcacttggg
     2761 ctttttttct tccccctctt tctttaaaga actgctcaat attcaatctg ttgtgaagaa
     2821 cctgatttgc actctgtagt gtttaaagaa acaaagaaac tctaatattg aatctcttaa
     2881 atttagtgta tgtaaacagc ttacaaatac gtattgtcta aatgcattta aatctgtttt
     2941 attcaaagaa aagctaaagc aaaaacactg gcatatgacc atgcaagact gtcagtgcca
     3001 acaaagacaa cactaatcag cacatcgtac actggattgc agtgcttccc agattattga
     3061 aaaatgttaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3121 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3181 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3241 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3301 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3361 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3421 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3481 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3541 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3601 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3661 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3721 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3781 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3841 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3901 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3961 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4021 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4081 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4141 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4201 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4261 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4321 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4381 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4441 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4501 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4561 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4621 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4681 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4741 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4801 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4861 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4921 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4981 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5041 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5101 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5161 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5221 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     5281 aataggcgta tcacgaggcc ctttcgtc
//
