LOCUS       sequence-452114-        5194 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold insertion into the N-terminus of the 
            ACTB locus. For actin visualization. To be co-transfected with sgRNA
            plasmid px330-PITCh-ACTB (Addgene #207748).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5194)
  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 5194)
  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-ACTB
FEATURES             Location/Qualifiers
     source          1..5194
                     /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             1410..2087
                     /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(2973..2989)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2973..2989)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2986..3008)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2997..3013
                     /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(3021..3027,3028..3045,3046..3051))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3066..3087
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3204..3221)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3375..3963)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3455..3474)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4134..4925,4926..4994))
                     /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     4757..4776
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4995..5099)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5167..5185
                     /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 ccctataaaa cccagcggcg
      421 cgacgcgcca ccaccgccga gaccgcgtcc gccccgcgag cacagagcct cgcctttgcc
      481 gatccgccgc ccgtccacac ccgccgccag gtaagcccgg ccagccgacc ggggcaggcg
      541 gctcacggcc cggccgcagg cggccgcggc cccttcgccc gtgcagagcc gccgtctggg
      601 ccgcagcggg gggcgcatgg ggggggaacc ggaccgccgt ggggggcgcg ggagaagccc
      661 ctgggcctcc ggagatgggg gacaccccac gccagttcgg aggcgcgagg ccgcgctcgg
      721 gaggcgcgct ccgggggtgc cgctctcggg gcgggggcaa ccggcggggt ctttgtctga
      781 gccgggctct tgccaatggg gatcgcaggg tgggcgcggc ggagcccccg ccaggcccgg
      841 tgggggctgg ggcgccattg cgcgtgcgcg ctggtccttt gggcgctaac tgcgtgcgcg
      901 ctgggaattg gcgctaattg cgcgtgcgcg ctgggactca aggcgctaac tgcgcgtgcg
      961 ttctggggcc cggggtgccg cggcctgggc tggggcgaag gcgggctcgg ccggaagggg
     1021 tggggtcgcc gcggctcccg ggcgcttgcg cgcacttcct gcccgagccg ctggccgccc
     1081 gagggtgtgg ccgctgcgtg cgcgcgcgcc gacccggcgc tgtttgaacc gggcggaggc
     1141 ggggctggcg cccggttggg agggggttgg ggcctggctt cctgccgcgc gccgcgggga
     1201 cgcctccgac cagtgtttgc cttttatggt aataacgcgg ccggcccggc ttcctttgtc
     1261 cccaatctgg gcgcgcgccg gcgccccctg gcggcctaag gactcggcgc gccggaagtg
     1321 gccagggcgg gggcgacctc ggctcacagc gcgcccggct attctcgcaa ctgacaatgg
     1381 gcaagagcaa gaccggtagc ggcggaagca tggtgtctac aggcgaggaa ctgtttaccg
     1441 gcgtggtgcc cttcaagttc cagctgaagg gcaccatcaa cggcaagagc ttcaccgtgg
     1501 aaggcgaggg cgagggcaat agccacgagg gcagccacaa aggcaagtat gtgtgcacca
     1561 gcggcaaact gccaatgtct tgggccgccc tgggaactag cttcggctat ggcatgaaat
     1621 actataccaa gtaccccagc ggcctgaaaa actggttcca cgaggtgatg cctgagggct
     1681 tcacctacga cagacacatc cagtacaagg gcgacggcag catccacgcc aagcaccagc
     1741 acttcatgaa gaacggcacc taccacaaca tcgtggagtt caccggccag gacttcaagg
     1801 agaacagccc cgtgctgacc ggcgacatgg acgtgagcct gcccaacgag gtgcagcaca
     1861 tccccattga tgacggcgtg gagtgcacag tgaccctgca gtaccctctg ctgagcgacg
     1921 aaagcaagtg cgtggaagcc tatcagaaca ccatcatcaa gcccctgcac aatcagccag
     1981 cccccgatgt gccatttcac tggatcagaa agcagtacac ccagagcaag gacgacaccg
     2041 aggagagaga ccacatcatc cagagcgaga ccctggaggc ccacctgcct tggcatgagc
     2101 ctagcgcaag cgcagtggga ggttccggag gaagcggagg atctggcggt accgatgatg
     2161 atatcgcagc gctcgtcgtc gacaacggct ccggcatgtg caaggccggc ttcgcgggcg
     2221 acgatgcccc ccgggccgtc ttcccctcca tcgtggggcg ccccaggcac caggtagggg
     2281 agctggctgg gtggggcagc cccgggagcg ggcgggaggc aagggcgctt tctctgcaca
     2341 ggagcctccc ggtttccggg gtgggggctg cgcccgtgct cagggcttct tgtcctttcc
     2401 ttcccagggc gtgatggtgg gcatgggtca gaaggattcc tatgtgggcg acgaggccca
     2461 gagcaagaga ggcatcctca ccctgaagta ccccatcgag cacggcatcg tcaccaactg
     2521 ggacgacatg gagaaaatct ggcaccacac cttctacaat gagctgcgtg tggctcccga
     2581 ggagcacccc gtgctgctga ccgaggcccc cctgaacccc aaggccaacc gcgagaagat
     2641 gacccaggtg agtggcccgc tacctcttct ggtggccgcc tccctccttc ctggcctccc
     2701 ggagctgcgc cctttctcac tggttctctc ttctgccgtt ttccgtagga ctctcttctc
     2761 tgacctgagt ctcctttgga actctgcagg ttctatttgc tttttcccag atgagctctt
     2821 tttctggtgt ttgtctctct gactaggtgt ctaagacagt gttgtgggtg taggtactaa
     2881 cactggctcg tgtgacaagg ccatgaggct ggtgtaaagc ggccttggag tgtgtattaa
     2941 gtaggcgcac agtaaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3001 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3061 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3121 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3181 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3241 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3301 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3361 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3421 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3481 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3541 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3601 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3661 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3721 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3781 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     3841 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3901 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3961 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4021 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4081 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4141 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4201 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4261 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4321 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4381 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4441 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4501 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4561 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4621 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4681 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4741 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4801 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4861 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4921 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4981 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5041 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5101 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     5161 tataaaaata ggcgtatcac gaggcccttt cgtc
//
