LOCUS       sequence-452117-        4510 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Donor template for 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 4510)
  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 4510)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-mStayGold-TUBA1B
FEATURES             Location/Qualifiers
     source          1..4510
                     /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..1667
                     /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(2289..2305)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2289..2305)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2302..2324)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2313..2329
                     /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(2337..2343,2344..2361,2362..2367))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2382..2403
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2520..2537)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2691..3279)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2771..2790)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3450..4241,4242..4310))
                     /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     4073..4092
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4311..4415)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4483..4501
                     /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 tggtgtctac aggcgaggaa ctgtttaccg
     1021 gcgtggtgcc cttcaagttc cagctgaagg gcaccatcaa cggcaagagc ttcaccgtgg
     1081 aaggcgaggg cgagggcaat agccacgagg gcagccacaa aggcaagtat gtgtgcacca
     1141 gcggcaaact gccaatgtct tgggccgccc tgggaactag cttcggctat ggcatgaaat
     1201 actataccaa gtaccccagc ggcctgaaaa actggttcca cgaggtgatg cctgagggct
     1261 tcacctacga cagacacatc cagtacaagg gcgacggcag catccacgcc aagcaccagc
     1321 acttcatgaa gaacggcacc taccacaaca tcgtggagtt caccggccag gacttcaagg
     1381 agaacagccc cgtgctgacc ggcgacatgg acgtgagcct gcccaacgag gtgcagcaca
     1441 tccccattga tgacggcgtg gagtgcacag tgaccctgca gtaccctctg ctgagcgacg
     1501 aaagcaagtg cgtggaagcc tatcagaaca ccatcatcaa gcccctgcac aatcagccag
     1561 cccccgatgt gccatttcac tggatcagaa agcagtacac ccagagcaag gacgacaccg
     1621 aggagagaga ccacatcatc cagagcgaga ccctggaggc ccacctgcct tggcatgagc
     1681 ctagcgcaag cgcagtgtcc ggactcagat ctcgagtgcg tgagtgcatc tccatccacg
     1741 ttggccaggc tggtgtccag attggcaatg cctgctggga gctctactgc ctggaacacg
     1801 gcatccagcc cgatggccag atgccaagtg acaagaccat tgggggagga gatgactcct
     1861 tcaacacctt cttcagtgag acgggcgctg gcaagcacgt gccccgggct gtgtttgtag
     1921 acttggaacc cacagtcatt ggtgagttga cctcagtaac ctgagatccc aggatgctgg
     1981 gacaggaggt ctgtccaggg gcttctcttg tcactcactc actccctccg tccttctctc
     2041 cctcctccag atgaagttcg cactggcacc taccgccagc tcttccaccc tgagcagctc
     2101 atcacaggca aggaagatgc tgccaataac tatgcccgag ggcactacac cattggcaag
     2161 gagatcattg accttgtgtt ggaccgaatt cgcaagctgg taagcaccac atataaatat
     2221 gcatttaatg tggtgtgata gttccagtgc aagttgggtg gagtgactga aagcttggcg
     2281 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     2341 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     2401 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2461 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2521 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2581 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2641 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2701 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2761 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2821 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2881 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2941 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3001 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3061 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3121 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3181 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3241 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3301 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3361 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3421 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3481 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3541 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3601 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3661 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3721 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3781 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3841 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3901 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3961 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4021 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4081 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4141 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4201 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4261 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4321 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4381 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4441 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     4501 ccctttcgtc
//
