LOCUS       sequence-452043-        4388 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold insertion into the C-terminus of the 
            ARL13B locus. For cilia visualization. To be co-transfected with 
            sgRNA plasmid pX330-PITCh-ARL13B (Addgene #227276).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4388)
  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 4388)
  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-C-Solo-mStayGold-ARL13B
FEATURES             Location/Qualifiers
     source          1..4388
                     /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             938..1615
                     /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(2167..2183)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2167..2183)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2180..2202)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2191..2207
                     /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(2215..2221,2222..2239,2240..2245))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2260..2281
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2398..2415)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2569..3157)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2649..2668)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3328..4119,4120..4188))
                     /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     3951..3970
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4189..4293)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4361..4379
                     /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 cacatttttt gttgcaatgt
      421 aaagtacaaa cttgatagtg gctatatttg gggtaagaat ccaaatttgg gggctattgc
      481 agtagtcaaa gtaagaagta aggaatggaa attggattgg agagtataac ataaggaagg
      541 aatattttaa ccattataat aagtaatatg tgattaatgc atgtgtggat tgaaggcaaa
      601 aggaatagag tataatttgg ctcaacagga tggaatgttg gtgccatctc ttaagattta
      661 aaatgcacga gagagaagga aatcttttag agaggcatgt caagattctg taagtttatc
      721 tctttggaaa tgtctaaaat gtctaaagtc taaaaaataa tgaactgtgt caaaaatctt
      781 gatgtaccat aactgttttg tgcatttggt tttctttctt ttcttagatt tctataggaa
      841 gccactgcct cccctggctg tgccacagcg acctaacagt gatgctcatg atgtgatctc
      901 aggaggaagc ggaggatctg gcggtaccgg cggaagcatg gtgtctacag gcgaggaact
      961 gtttaccggc gtggtgccct tcaagttcca gctgaagggc accatcaacg gcaagagctt
     1021 caccgtggaa ggcgagggcg agggcaatag ccacgagggc agccacaaag gcaagtatgt
     1081 gtgcaccagc ggcaaactgc caatgtcttg ggccgccctg ggaactagct tcggctatgg
     1141 catgaaatac tataccaagt accccagcgg cctgaaaaac tggttccacg aggtgatgcc
     1201 tgagggcttc acctacgaca gacacatcca gtacaagggc gacggcagca tccacgccaa
     1261 gcaccagcac ttcatgaaga acggcaccta ccacaacatc gtggagttca ccggccagga
     1321 cttcaaggag aacagccccg tgctgaccgg cgacatggac gtgagcctgc ccaacgaggt
     1381 gcagcacatc cccattgatg acggcgtgga gtgcacagtg accctgcagt accctctgct
     1441 gagcgacgaa agcaagtgcg tggaagccta tcagaacacc atcatcaagc ccctgcacaa
     1501 tcagccagcc cccgatgtgc catttcactg gatcagaaag cagtacaccc agagcaagga
     1561 cgacaccgag gagagagacc acatcatcca gagcgagacc ctggaggccc acctgggagg
     1621 ttccctcgag accggtaaga gcaagtgata aacaagacgt atcgaagagt tctcttaata
     1681 tcagcaaggt gaactgggac attcttcttt ctcagaagaa gaaacatctt gtaaattgat
     1741 gactggggca agataaccat aataatttta gtgagaagat taatactcaa ggacctgact
     1801 tgataattac ttatttgtgt ttttcatggt taaaaaaata aaagaagcac aatgaccagt
     1861 acatgaaatc agcatttgga ccaaattagc aagatttact gttgactctg gtttatacat
     1921 ccccactcat gagcatactt ctgaaggaaa actttacaaa aagagccaat ggactcagca
     1981 ctttctttac tatttgttca atagcctata tttctagata ttaaatattt tttgtaagat
     2041 atatgcacat agaaggggga cttctaggaa tttataacca aaattttaaa actattttta
     2101 tattttttta aatctttaaa aattttaatt actgtgatat tgattataaa gcttggcgta
     2161 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2221 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2281 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2341 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2401 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2461 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2521 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2581 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2641 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2701 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2761 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2821 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2881 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2941 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3001 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3061 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3121 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3181 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3241 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3301 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3361 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3421 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     3481 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3541 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3601 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     3661 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     3721 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3781 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3841 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     3901 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     3961 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4021 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4081 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4141 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4201 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4261 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4321 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     4381 ctttcgtc
//
