LOCUS       sequence-413790-        4501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mScarlet 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 4501)
  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 4501)
  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-mScarlet-TUBA1B
FEATURES             Location/Qualifiers
     source          1..4501
                     /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..1685
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1123..1141)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1391..1410
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2280..2296)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2280..2296)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2293..2315)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2304..2320
                     /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(2328..2334,2335..2352,2353..2358))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2373..2394
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2511..2528)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2682..3270)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2762..2781)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3441..4232,4233..4301))
                     /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     4064..4083
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4302..4406)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4474..4492
                     /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 tggtgagcaa gggcgaggca gtgatcaagg
     1021 agttcatgcg gttcaaggtg cacatggagg gctccatgaa cggccacgag ttcgagatcg
     1081 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     1141 agggtggccc cctgcccttc tcctgggaca tcctgtcccc tcagttcatg tacggctcca
     1201 gggccttcac caagcacccc gccgacatcc ccgactacta taagcagtcc ttccccgagg
     1261 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgccgtgacc gtgacccagg
     1321 acacctccct ggaggacggc accctgatct acaaggtgaa gctccgcggc accaacttcc
     1381 ctcctgacgg ccccgtaatg cagaagaaga caatgggctg ggaagcgtcc accgagcggt
     1441 tgtaccccga ggacggcgtg ctgaagggcg acattaagat ggccctgcgc ctgaaggacg
     1501 gcggccgcta cctggcggac ttcaagacca cctacaaggc caagaagccc gtgcagatgc
     1561 ccggcgccta caacgtcgac cgcaagttgg acatcacctc ccacaacgag gactacaccg
     1621 tggtggaaca gtacgaacgc tccgagggcc gccactccac cggcggcatg gacgagctgt
     1681 acaagtccgg actcagatct cgagtgggta ccgagtgcat ctccatccac gttggccagg
     1741 ctggtgtcca gattggcaat gcctgctggg agctctactg cctggaacac ggcatccagc
     1801 ccgatggcca gatgccaagt gacaagacca ttgggggagg agatgactcc ttcaacacct
     1861 tcttcagtga gacgggcgct ggcaagcacg tgccccgggc tgtgtttgta gacttggaac
     1921 ccacagtcat tggtgagttg acctcagtaa cctgagatcc caggatgctg ggacaggagg
     1981 tctgtccagg ggcttctctt gtcactcact cactccctcc gtccttctct ccctcctcca
     2041 gatgaagttc gcactggcac ctaccgccag ctcttccacc ctgagcagct catcacaggc
     2101 aaggaagatg ctgccaataa ctatgcccga gggcactaca ccattggcaa ggagatcatt
     2161 gaccttgtgt tggaccgaat tcgcaagctg gtaagcacca catataaata tgcatttaat
     2221 gtggtgtgat agttccagtg caagttgggt ggagtgactg aaagcttggc gtaatcatgg
     2281 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2341 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2401 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2461 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2521 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2581 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2641 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2701 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2761 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2821 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2881 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2941 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3001 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3061 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3121 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3181 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3241 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3301 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3361 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3421 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3481 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3541 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3601 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3661 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3721 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3781 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3841 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3901 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3961 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4021 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4081 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4141 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4201 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4261 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4321 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4381 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     4441 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     4501 c
//
