LOCUS       sequence-451732-        5547 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold-EFS-Blast insertion into the C-terminus
            of the ARL13B locus. For cilia visualization. To be co-transfected 
            with plasmid pX330-PITCh-ARL13B (Addgene #227276).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5547)
  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 5547)
  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-mStayGold-EFS-Blast-ARL13B
FEATURES             Location/Qualifiers
     source          1..5547
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..33
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(71..93)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     193..212
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     406..428
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     420..437
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     421..437
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1035..1712
                     /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"
     CDS             1728..1769
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1791..1824)
                     /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)."
     polyA_signal    1946..2067
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1983..2002)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2037..2056
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2117..2328
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2333..2342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2339..2734
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(2741..2774)
                     /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)."
     protein_bind    3392..3408
                     /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(3416..3422,3423..3440,3441..3446))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3461..3482
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3599..3616)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3770..4358)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3850..3869)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4529..5320,5321..5389))
                     /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     5152..5171
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5390..5494)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
       61 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
      121 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
      181 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
      241 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc
      301 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
      361 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
      421 gtaaaacgac ggccagtgaa ttcgctttta tttttaaaag ggaaaatgct attatatttg
      481 gtgcttagaa aaacattttt tgttgcaatg taaagtacaa acttgatagt ggctatattt
      541 ggggtaagaa tccaaatttg ggggctattg cagtagtcaa agtaagaagt aaggaatgga
      601 aattggattg gagagtataa cataaggaag gaatatttta accattataa taagtaatat
      661 gtgattaatg catgtgtgga ttgaaggcaa aaggaataga gtataatttg gctcaacagg
      721 atggaatgtt ggtgccatct cttaagattt aaaatgcacg agagagaagg aaatctttta
      781 gagaggcatg tcaagattct gtaagtttat ctctttggaa atgtctaaaa tgtctaaagt
      841 ctaaaaaata atgaactgtg tcaaaaatct tgatgtacca taactgtttt gtgcatttgg
      901 ttttctttct tttcttagat ttctatagga agccactgcc tcccctggct gtgccacagc
      961 gacctaacag tgatgctcat gatgtgatct caggaggaag cggaggatct ggcggtaccg
     1021 gaggtggcgg aagcatggtg tctacaggcg aggaactgtt taccggcgtg gtgcccttca
     1081 agttccagct gaagggcacc atcaacggca agagcttcac cgtggaaggc gagggcgagg
     1141 gcaatagcca cgagggcagc cacaaaggca agtatgtgtg caccagcggc aaactgccaa
     1201 tgtcttgggc cgccctggga actagcttcg gctatggcat gaaatactat accaagtacc
     1261 ccagcggcct gaaaaactgg ttccacgagg tgatgcctga gggcttcacc tacgacagac
     1321 acatccagta caagggcgac ggcagcatcc acgccaagca ccagcacttc atgaagaacg
     1381 gcacctacca caacatcgtg gagttcaccg gccaggactt caaggagaac agccccgtgc
     1441 tgaccggcga catggacgtg agcctgccca acgaggtgca gcacatcccc attgatgacg
     1501 gcgtggagtg cacagtgacc ctgcagtacc ctctgctgag cgacgaaagc aagtgcgtgg
     1561 aagcctatca gaacaccatc atcaagcccc tgcacaatca gccagccccc gatgtgccat
     1621 ttcactggat cagaaagcag tacacccaga gcaaggacga caccgaggag agagaccaca
     1681 tcatccagag cgagaccctg gaggcccacc tgggaggtgg cggttccggc aagcccatcc
     1741 ccaaccccct gctgggcctg gacagcaccg gaggtggcgg atcaacgcgt taccgttcgt
     1801 atagcataca ttatacgaag ttatctggat cctgataatg atcataatca gccataccac
     1861 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     1921 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     1981 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2041 tttgtccaaa ctcatcaatg tatcttatct agataggtct tgaaaggagt gggaattggc
     2101 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     2161 ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     2221 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     2281 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggg ccgccaccat
     2341 ggccaagcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg ctacaatcaa
     2401 cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta gcgacggccg
     2461 catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag aactcgtggt
     2521 gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg cgatcggaaa
     2581 tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc tcgatctgca
     2641 tcctgggatc aaagccatag tgaaggacag tgatggacag ccgacggcag ttgggattcg
     2701 tgaattgctg ccctctggtt atgtgtggga gggcgctagc ataacttcgt atagcataca
     2761 ttatacgaac ggtagcctcg agaccggtaa gagcaagtga taaacaagac gtatcgaaga
     2821 gttctcttaa tatcagcaag gtgaactggg acattcttct ttctcagaag aagaaacatc
     2881 ttgtaaattg atgactgggg caagataacc ataataattt tagtgagaag attaatactc
     2941 aaggacctga cttgataatt acttatttgt gtttttcatg gttaaaaaaa taaaagaagc
     3001 acaatgacca gtacatgaaa tcagcatttg gaccaaatta gcaagattta ctgttgactc
     3061 tggtttatac atccccactc atgagcatac ttctgaagga aaactttaca aaaagagcca
     3121 atggactcag cactttcttt actatttgtt caatagccta tatttctaga tattaaatat
     3181 tttttgtaag atatatgcac atagaagggg gacttctagg aatttataac caaaatttta
     3241 aaactatttt tatatttttt taaatcttta aaaattttaa ttactgtgat attgattata
     3301 cacctttttt atagatgatt tgtttaaatt atatctggaa aatagagtta agcttggcgt
     3361 aatcatggtc atagctgttt actgtgtgaa attgttatcc gctcacaatt ccacacaaca
     3421 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3481 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3541 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3601 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3661 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3721 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3781 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3841 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3901 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3961 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4021 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4081 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4141 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4201 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4261 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4321 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4381 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4441 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4501 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4561 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4621 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4681 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4741 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4801 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4861 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4921 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4981 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5041 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5101 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5161 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5221 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5281 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5341 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5401 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5461 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5521 acgtctaaga aaccattatt atcatga
//
