LOCUS       sequence-413793-        5338 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Puro-2A-moxGFP 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 5338)
  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 5338)
  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-moxGFP-TUBA1B
FEATURES             Location/Qualifiers
     source          1..5338
                     /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             1734..1760
                     /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             1734..1760
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1764..1790
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1800..1802,1803..1805,1806..2516)
                     /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(1845..1866)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2453..2474
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3117..3133)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3117..3133)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3130..3152)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3141..3157
                     /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(3165..3171,3172..3189,3190..3195))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3210..3231
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3348..3365)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3519..4107)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3599..3618)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4278..5069,5070..5138))
                     /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     4901..4920
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5139..5243)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5311..5329
                     /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 cgttacccct
     1741 acgacgtgcc cgactacgcc ggctatccgt atgatgtccc ggactatgca ggaagcggaa
     1801 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1861 gcgacgtgaa cggccacaag ttctccgtgc ggggcgaggg cgagggcgat gccaccaacg
     1921 gcaagctgac cctgaagttc atcagcacca ccggcaagct gcccgtgccc tggcccaccc
     1981 tcgtgaccac cctgacctac ggcgtgcaga gcttctcccg ctaccccgac cacatgaagc
     2041 gccacgactt cttcaagagc gccatgcccg aaggctacgt ccaggagcgc accatctcct
     2101 tcaaggacga cggcacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2161 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2221 agctggagta caacttcaac tcccacaacg tctatatcac cgccgacaag cagaagaacg
     2281 gcatcaaggc caacttcaag atccgccata acgtggagga cggctccgtg cagctcgccg
     2341 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2401 acctgtccac ccagtccaag ctgtccaaag accccaacga gaagcgcgat cacatggtcc
     2461 tgctggagtt cgtgaccgcc gccgggatca ctcacggcat ggacgagctg tacaagctcg
     2521 agggaggaag cggaggatct ggcggtaccg agtgcatctc catccacgtt ggccaggctg
     2581 gtgtccagat tggcaatgcc tgctgggagc tctactgcct ggaacacggc atccagcccg
     2641 atggccagat gccaagtgac aagaccattg ggggaggaga tgactccttc aacaccttct
     2701 tcagtgagac gggcgctggc aagcacgtgc cccgggctgt gtttgtagac ttggaaccca
     2761 cagtcattgg tgagttgacc tcagtaacct gagatcccag gatgctggga caggaggtct
     2821 gtccaggggc ttctcttgtc actcactcac tccctccgtc cttctctccc tcctccagat
     2881 gaagttcgca ctggcaccta ccgccagctc ttccaccctg agcagctcat cacaggcaag
     2941 gaagatgctg ccaataacta tgcccgaggg cactacacca ttggcaagga gatcattgac
     3001 cttgtgttgg accgaattcg caagctggta agcaccacat ataaatatgc atttaatgtg
     3061 gtgtgatagt tccagtgcaa gttgggtgga gtgactgaaa gcttggcgta atcatggtca
     3121 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3181 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3241 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3301 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3361 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3421 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3481 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3541 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3601 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     3661 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     3721 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     3781 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     3841 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     3901 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     3961 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4021 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4081 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4141 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4201 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4261 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4321 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4381 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4441 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4501 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4561 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     4621 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4681 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4741 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4801 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4861 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4921 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4981 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5041 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5101 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5161 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5221 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     5281 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
