LOCUS       sequence-413791-        4537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for sTagRFP 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 4537)
  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 4537)
  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-Solo-sTagRFP-TUBA1B
FEATURES             Location/Qualifiers
     source          1..4537
                     /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             990..1721
                     /codon_start=1
                     /product="photostable variant of TagRFP (Shaner et al., 
                     2008)"
                     /label=TagRFP-T
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTV
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     primer_bind     complement(2316..2332)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2316..2332)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2329..2351)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2340..2356
                     /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(2364..2370,2371..2388,2389..2394))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2409..2430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2547..2564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2718..3306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2798..2817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3477..4268,4269..4337))
                     /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     4100..4119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4338..4442)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4510..4528
                     /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 ggaggttcca tggtgtctaa gggcgaggaa ctgattaagg
     1021 agaatatgca catgaagctg tacatggagg gcaccgtgaa caaccaccac ttcaaatgca
     1081 cctccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg
     1141 agggcggccc tctccccttc gccttcgaca tcctggctac cagcttcatg tacggcagca
     1201 gaaccttcat caaccacacc cagggcatcc ccgatttctt taagcagtcc ttcccagagg
     1261 gcttcacatg ggagagagtc accacatacg aggacggggg cgtgctgacc gccacccagg
     1321 acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc
     1381 catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccaac accgagatgc
     1441 tgtaccccgc tgacggcggc ctggaaggca gaaccgtcat ggccctgaag ctcgtgggcg
     1501 ggggccacct gatctgcaac ttcaagacca catacaggtc caagaaaccc gctaagaacc
     1561 tgaagatgcc cggagtgtac tatgtggacc acagactgga gagaatcaag gaggccgaca
     1621 aagagacata cgtcgagcag cacgaggtgg ctgtggccag atactgcgac ctccctagca
     1681 aactgggcca caagctgaac ggcatggacg agctgtacaa gtccggactc agatctcgag
     1741 tgggtaccga gtgcatctcc atccacgttg gccaggctgg tgtccagatt ggcaatgcct
     1801 gctgggagct ctactgcctg gaacacggca tccagcccga tggccagatg ccaagtgaca
     1861 agaccattgg gggaggagat gactccttca acaccttctt cagtgagacg ggcgctggca
     1921 agcacgtgcc ccgggctgtg tttgtagact tggaacccac agtcattggt gagttgacct
     1981 cagtaacctg agatcccagg atgctgggac aggaggtctg tccaggggct tctcttgtca
     2041 ctcactcact ccctccgtcc ttctctccct cctccagatg aagttcgcac tggcacctac
     2101 cgccagctct tccaccctga gcagctcatc acaggcaagg aagatgctgc caataactat
     2161 gcccgagggc actacaccat tggcaaggag atcattgacc ttgtgttgga ccgaattcgc
     2221 aagctggtaa gcaccacata taaatatgca tttaatgtgg tgtgatagtt ccagtgcaag
     2281 ttgggtggag tgactgaaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     2341 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     2401 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     2461 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2521 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2581 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2641 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2701 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2761 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2821 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2881 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2941 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3001 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3061 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3121 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3181 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3241 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3301 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3361 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3421 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3481 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3541 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3601 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3661 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3721 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3781 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3841 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3901 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3961 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4021 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4081 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4141 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4201 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4261 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4321 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4381 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4441 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     4501 acctataaaa ataggcgtat cacgaggccc tttcgtc
//
