LOCUS       sequence-452073-        4486 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold insertion into the C-terminus of the 
            VIM locus. For intermediate filament visualization. To be 
            co-transfected with sgRNA plasmid px330-PITCh-VIM (Addgene #227301).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4486)
  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 4486)
  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-VIM
FEATURES             Location/Qualifiers
     source          1..4486
                     /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             987..1664
                     /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(2265..2281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2265..2281)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2278..2300)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2289..2305
                     /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(2313..2319,2320..2337,2338..2343))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2358..2379
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2496..2513)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2667..3255)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2747..2766)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3426..4217,4218..4286))
                     /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     4049..4068
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4287..4391)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4459..4477
                     /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 cgatcctgga catatggtgt
      421 cttcctacct ccatacttta tagattccta aacccatcta tataatacaa gcatgtgcca
      481 tacgatcatt tagtttctta ttacctccct atgccaggaa agaaatagtt gcaatttatt
      541 gtagtcatca tgaaatcttc ccttgcacat aaatttaaaa tgtacctgct gcacatttta
      601 atatgtctta attgctttta aacttggctg tattgtgtac aactattata ccatctttta
      661 taaacacagt tttttaagaa atttcttttt gtaagttaca acattccact ggatccttat
      721 attgcctgta gtggaagagg gtcttgtgtg tctgcccctt ctagttttca ctcatgcaga
      781 agcaacataa ccttctgatt tgcacaataa attacatata tttagcagga tttttatttg
      841 ccgtgatata taggataatt tagtctttgg catgtggcat tatatttatt ttggtttttt
      901 tttttaaaca ggttatcaac gaaacttctc agcatcacga tgaccttgaa ggaggaagcg
      961 gaggatctgg cggtaccggc ggaagcatgg tgtctacagg cgaggaactg tttaccggcg
     1021 tggtgccctt caagttccag ctgaagggca ccatcaacgg caagagcttc accgtggaag
     1081 gcgagggcga gggcaatagc cacgagggca gccacaaagg caagtatgtg tgcaccagcg
     1141 gcaaactgcc aatgtcttgg gccgccctgg gaactagctt cggctatggc atgaaatact
     1201 ataccaagta ccccagcggc ctgaaaaact ggttccacga ggtgatgcct gagggcttca
     1261 cctacgacag acacatccag tacaagggcg acggcagcat ccacgccaag caccagcact
     1321 tcatgaagaa cggcacctac cacaacatcg tggagttcac cggccaggac ttcaaggaga
     1381 acagccccgt gctgaccggc gacatggacg tgagcctgcc caacgaggtg cagcacatcc
     1441 ccattgatga cggcgtggag tgcacagtga ccctgcagta ccctctgctg agcgacgaaa
     1501 gcaagtgcgt ggaagcctat cagaacacca tcatcaagcc cctgcacaat cagccagccc
     1561 ccgatgtgcc atttcactgg atcagaaagc agtacaccca gagcaaggac gacaccgagg
     1621 agagagacca catcatccag agcgagaccc tggaggccca cctgggaggt tccctcgaga
     1681 ccggtaagag caagtgataa aaattgcaca cactcagtgc agcaatatat taccagcaag
     1741 aataaaaaag aaatccatat cttaaagaaa cagctttcaa gtgcctttct gcagtttttc
     1801 aggagcgcaa gatagatttg gaatatgaat aagctctagt tcttaacaac cgacactcct
     1861 acaagattta gaaaaaagtt tacaacataa tctagtttac agaaaaatct tgtgctagaa
     1921 tactttttaa aaggtatctt gaataccatt aaaactgctt ttttttttcc agcaagtatc
     1981 caaccaactt ggttctgctt caataaatct ttggaaaaac tcttttgttg tgttatttat
     2041 tggataatat ctaaacaatt ctctacttgg tcctattagt taatttgtca ttacaatcat
     2101 gtaagttgat aaattcaggt tatttatgct tgagatgtag ttcttaattt tgtcattttt
     2161 gatagacctc acttcttttt attattactt aaaaacattt acaaataggt ggtgtcgaat
     2221 aaaataactt gtaccaaaat gaagataagc ttggcgtaat catggtcata gctgtttcct
     2281 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     2341 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     2401 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2461 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2521 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2581 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2641 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2701 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2761 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2821 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2881 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2941 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3001 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3061 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3121 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3181 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3241 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3301 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3361 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3421 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3481 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3541 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3601 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3661 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3721 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3781 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3841 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3901 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3961 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     4021 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     4081 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     4141 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     4201 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4261 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4321 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4381 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     4441 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
