LOCUS       sequence-452094-        5350 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Puro-2A-mStayGold insertion into the N-terminus 
            of the TUBA1B locus. For tubulin visualization. To be co-transfected
            with sgRNA plasmid px330-PITCh-TUBA1B (Addgene #207763).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5350)
  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 5350)
  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-N-Puro-mStayGold-TUBA1B
FEATURES             Location/Qualifiers
     source          1..5350
                     /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"
     protein_bind    complement(981..1014)
                     /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             1023..1619
                     /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(1023..1042)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1519..1539
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1629..1685
                     /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"
     protein_bind    complement(1692..1725)
                     /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)."
     CDS             1749..1790
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1806..2483
                     /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(3129..3145)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3129..3145)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3142..3164)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3153..3169
                     /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(3177..3183,3184..3201,3202..3207))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3222..3243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3360..3377)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3531..4119)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3611..3630)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4290..5081,5082..5150))
                     /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     4913..4932
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5151..5255)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5323..5341
                     /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 cctgcccgga aatgtgacca
      421 cctagtctaa agtgggcttc tggggcctga gcgctggatg gatgcccacc ttcctgtctt
      481 ggtcctccaa aggaggaagc tgtgactgag ctgtcttggt ctggaaggag gccttcccgg
      541 tttaggatgg gaaggtaaca ttcattaaaa gcaacgtaga ctatagtgta gctgttctca
      601 aaagtagtac atcttagaaa aggatcttta gaaaagatcg ctttagaaaa ggaaattcgt
      661 tttcagatta cgtgagtagc ctaggtaaca cagccagacc tcatctccac aaaaaaaatg
      721 aaaaaattag ccagcttggt ggtctgtgcc tgtggtccca gctgctccag aggctgaggt
      781 ggggggatga ctggagccta ggctgcagtg agcctagatg gcatcactgc actcaagact
      841 gggcgacaga ccttatctct aaaaaaataa agattgcatg agtattttgt tccacttgac
      901 agtcatcaat agattggttt aaattgtgat atctttttta cttcccgcag agagaatgtg
      961 gcaagagcaa gaccggtagc taccgttcgt atagcataca ttatacgaag ttatctgcta
     1021 gcatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     1081 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     1141 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     1201 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     1261 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     1321 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     1381 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     1441 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     1501 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     1561 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg
     1621 gatccggagc tactaacttc agcctgctga agcaggccgg agacgtggag gagaaccctg
     1681 gaccttctag aataacttcg tatagcatac attatacgaa cggtagcacg cgtggaggtg
     1741 gcggttccgg caagcccatc cccaaccccc tgctgggcct ggacagcacc ggaggtggcg
     1801 gaagcatggt gtctacaggc gaggaactgt ttaccggcgt ggtgcccttc aagttccagc
     1861 tgaagggcac catcaacggc aagagcttca ccgtggaagg cgagggcgag ggcaatagcc
     1921 acgagggcag ccacaaaggc aagtatgtgt gcaccagcgg caaactgcca atgtcttggg
     1981 ccgccctggg aactagcttc ggctatggca tgaaatacta taccaagtac cccagcggcc
     2041 tgaaaaactg gttccacgag gtgatgcctg agggcttcac ctacgacaga cacatccagt
     2101 acaagggcga cggcagcatc cacgccaagc accagcactt catgaagaac ggcacctacc
     2161 acaacatcgt ggagttcacc ggccaggact tcaaggagaa cagccccgtg ctgaccggcg
     2221 acatggacgt gagcctgccc aacgaggtgc agcacatccc cattgatgac ggcgtggagt
     2281 gcacagtgac cctgcagtac cctctgctga gcgacgaaag caagtgcgtg gaagcctatc
     2341 agaacaccat catcaagccc ctgcacaatc agccagcccc cgatgtgcca tttcactgga
     2401 tcagaaagca gtacacccag agcaaggacg acaccgagga gagagaccac atcatccaga
     2461 gcgagaccct ggaggcccac ctgccttggc atgagcctag cgcaagcgca gtgggaggtg
     2521 gcggttccct cgagggagga agcggaggat ctggcggtac cgagtgcatc tccatccacg
     2581 ttggccaggc tggtgtccag attggcaatg cctgctggga gctctactgc ctggaacacg
     2641 gcatccagcc cgatggccag atgccaagtg acaagaccat tgggggagga gatgactcct
     2701 tcaacacctt cttcagtgag acgggcgctg gcaagcacgt gccccgggct gtgtttgtag
     2761 acttggaacc cacagtcatt ggtgagttga cctcagtaac ctgagatccc aggatgctgg
     2821 gacaggaggt ctgtccaggg gcttctcttg tcactcactc actccctccg tccttctctc
     2881 cctcctccag atgaagttcg cactggcacc taccgccagc tcttccaccc tgagcagctc
     2941 atcacaggca aggaagatgc tgccaataac tatgcccgag ggcactacac cattggcaag
     3001 gagatcattg accttgtgtt ggaccgaatt cgcaagctgg taagcaccac atataaatat
     3061 gcatttaatg tggtgtgata gttccagtgc aagttgggtg gagtgactga aagcttggcg
     3121 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3181 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3241 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3301 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3361 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3421 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3481 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3541 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3601 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3661 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3721 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3781 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3841 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3901 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3961 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4021 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4081 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4141 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4201 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4261 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4321 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4381 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4441 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4501 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4561 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4621 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4681 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4741 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4801 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4861 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4921 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4981 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5041 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5101 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5161 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5221 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5281 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     5341 ccctttcgtc
//
