LOCUS       sequence-451297-        4515 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold insertion into the N-terminus of the 
            CENPA locus. For centromere visualization. To be co-transfected with
            sgRNA plasmid px330-CENPA (Addgene #227282).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4515)
  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 4515)
  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-mStayGold-CENPA
FEATURES             Location/Qualifiers
     source          1..4515
                     /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             982..1659
                     /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(2294..2310)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2294..2310)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2307..2329)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2318..2334
                     /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(2342..2348,2349..2366,2367..2372))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2387..2408
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2525..2542)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2696..3284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2776..2795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3455..4246,4247..4315))
                     /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     4078..4097
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4316..4420)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4488..4506
                     /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 cctaaggcaa agcataaaat
      421 gtttgaggga agcgactttt ctagaaagca cgttctacaa acacaccatc cacgttccaa
      481 actgagcttc ggtgttttga ctggagggca cctgaggacg tcagtacggt cccgcaaaga
      541 ccagcaacca gattggcaag cagggggcag gagaatttcc cgccccatct ctttagttgt
      601 ctattggttg attttaccat cagtctcccc tgtcctccta tcatcgccgc tatatttgtg
      661 tctcggtgca tactctcatt ggccctacct gcagtcgctc tacgtaaggg gcgttccaga
      721 gtcagccact gggagtcgct ggattcgggt tttaaaaaac gccggccgtg aagtgggcgg
      781 agcgagcgat ttgaacgcga gcggcgcgga cttctgccaa gcaccggctc atgtgaggct
      841 cgcggcacag cgttctctgg gctccccaga agccagcctt tcgctcccgg acccggcagc
      901 ccgagcagga gccgtgggac cgggcgccag caccctctgc ggcgtgtcat gggcaagagc
      961 aagaccggta gcggcggaag catggtgtct acaggcgagg aactgtttac cggcgtggtg
     1021 cccttcaagt tccagctgaa gggcaccatc aacggcaaga gcttcaccgt ggaaggcgag
     1081 ggcgagggca atagccacga gggcagccac aaaggcaagt atgtgtgcac cagcggcaaa
     1141 ctgccaatgt cttgggccgc cctgggaact agcttcggct atggcatgaa atactatacc
     1201 aagtacccca gcggcctgaa aaactggttc cacgaggtga tgcctgaggg cttcacctac
     1261 gacagacaca tccagtacaa gggcgacggc agcatccacg ccaagcacca gcacttcatg
     1321 aagaacggca cctaccacaa catcgtggag ttcaccggcc aggacttcaa ggagaacagc
     1381 cccgtgctga ccggcgacat ggacgtgagc ctgcccaacg aggtgcagca catccccatt
     1441 gatgacggcg tggagtgcac agtgaccctg cagtaccctc tgctgagcga cgaaagcaag
     1501 tgcgtggaag cctatcagaa caccatcatc aagcccctgc acaatcagcc agcccccgat
     1561 gtgccatttc actggatcag aaagcagtac acccagagca aggacgacac cgaggagaga
     1621 gaccacatca tccagagcga gaccctggag gcccacctgc cttggcatga gcctagcgca
     1681 agcgcagtgg gaggttccgg aggaagcgga ggatctggcg gtaccggccc gcgccgcaga
     1741 agccgaaagc ccgaggcccc gaggaggcgc agcccgagcc cgaccccgac ccccggcccc
     1801 tcccggcggg gcccctcctt aggtaaccgg ccgcggcccc atctcgaaga ggagaaaacc
     1861 gaggctcgca ggcggagccc ggatctcccg agtcccgatc ccgtggcact tccctgttgg
     1921 gggacaacgc ggttccgccg tccggcatcc gctccgggca cgggcggtgc cagccctgcg
     1981 aggggctgga gcaggcggtt ccgaaagcgg gaagggaccg gcttagccac ttggctccta
     2041 acgccgtctt cttgttagga ctagtggtta cccagcgccc accgcccacc gccaaccagg
     2101 cattgcctcc cactgttcgt gcgttatctc agcttcatat tgtgcccatt ttacagctaa
     2161 tgagaaagcc gaggcacagg ccagagagct tgagcagagt cacacaactt ggtgttctgg
     2221 cggacacggg atttccaatc ctctgtgttt gtgcattttt aaattgcaaa agtataagct
     2281 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2341 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2401 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2461 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2521 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2581 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2641 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2701 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2761 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2821 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2881 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2941 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3001 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3061 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3121 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3181 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3241 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3301 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3361 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3421 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3481 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3541 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3601 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3661 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3721 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3781 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3841 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3901 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3961 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4021 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4081 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4141 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4201 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4261 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4321 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4381 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4441 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4501 cgaggccctt tcgtc
//
