LOCUS       sequence-451726-        4815 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold insertion into the N-terminus of the 
            LMNB1 locus. For nuclear envelope visualization. To be 
            co-transfected with sgRNA plasmid px330-PITCh-LMNB1 (Addgene 
            #207770).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4815)
  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 4815)
  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-LMNB1
FEATURES             Location/Qualifiers
     source          1..4815
                     /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             1132..1809
                     /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(2594..2610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2594..2610)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2607..2629)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2618..2634
                     /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(2642..2648,2649..2666,2667..2672))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2687..2708
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2825..2842)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2996..3584)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3076..3095)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3755..4546,4547..4615))
                     /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     4378..4397
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4616..4720)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4788..4806
                     /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 cggggcggcg ggagggccct
      421 gggcccagag ccgcgcgggt gggcagtccc aggcgtcctt ccttacagcc ctgagcctgg
      481 tccgggaacc gcccagccgg gagggccgag ctgacggttg cccaagggcc agattttaaa
      541 tttacaggcc cggcccccga accgccgaag cgcgctgcct gctccccatt ggcccatggt
      601 agtcacgtgg aggcgccggg gcgtgccggc catgttgggg agtgcggcgc cgcggcccgc
      661 gccacctccg ccccccgcgg cttgcctcca gcccgcccct cccggccctc ctccccccgc
      721 ccgccgctcc gtgcagcctg agaggaaaca aagtgctgcg agcaggagac ggcggcggcg
      781 cgaaccctgc tgggcctcca ttcaccctcg tcttgcattt tcccgcgtgc gtgtgtgagt
      841 gggtgtgtgt gttttcttac aaagggtatt tcgcgatcga tcgattgatt cgtagttccc
      901 ccccgcgcgc ctttgccctt tgtgctgtaa tcgagctccc gccatcccag gtgcttctcc
      961 gttcctctaa acgccagcgt ctggacgtga gcgcaggtcg ccggtttgtg ccttcggtcc
     1021 ccgcttcgcc ccctgccgtc ccctccttat cacggtcccg ctcgcggcct cgccgccccg
     1081 ctgtctccgc cgcccgccat gggcaagagc aagaccggta gcggcggaag catggtgtct
     1141 acaggcgagg aactgtttac cggcgtggtg cccttcaagt tccagctgaa gggcaccatc
     1201 aacggcaaga gcttcaccgt ggaaggcgag ggcgagggca atagccacga gggcagccac
     1261 aaaggcaagt atgtgtgcac cagcggcaaa ctgccaatgt cttgggccgc cctgggaact
     1321 agcttcggct atggcatgaa atactatacc aagtacccca gcggcctgaa aaactggttc
     1381 cacgaggtga tgcctgaggg cttcacctac gacagacaca tccagtacaa gggcgacggc
     1441 agcatccacg ccaagcacca gcacttcatg aagaacggca cctaccacaa catcgtggag
     1501 ttcaccggcc aggacttcaa ggagaacagc cccgtgctga ccggcgacat ggacgtgagc
     1561 ctgcccaacg aggtgcagca catccccatt gatgacggcg tggagtgcac agtgaccctg
     1621 cagtaccctc tgctgagcga cgaaagcaag tgcgtggaag cctatcagaa caccatcatc
     1681 aagcccctgc acaatcagcc agcccccgat gtgccatttc actggatcag aaagcagtac
     1741 acccagagca aggacgacac cgaggagaga gaccacatca tccagagcga gaccctggag
     1801 gcccacctgc cttggcatga gcctagcgca agcgcagtgg gaggttccgg aggaagcgga
     1861 ggatctggcg gtaccgcgac tgcgaccccc gtgccgccgc ggatgggcag ccgcgctggc
     1921 ggccccacca cgccgctgag ccccacgcgc ctgtcgcggc tccaggagaa ggaggagctg
     1981 cgcgagctca atgaccggct ggcggtgtac atcgacaagg tgcgcagcct ggagacggag
     2041 aacagcgcgc tgcagctgca ggtgacggag cgcgaggagg tgcgcggccg tgagctcacc
     2101 ggcctcaagg cgctctacga gaccgagctg gccgacgcgc gacgcgcgct cgacgacacg
     2161 gcccgcgagc gcgccaagct gcagatcgag ctgggcaagt gcaaggcgga acacgaccag
     2221 ctgctcctca agtgagtgct agctggcggc cgcgttagcg ccaaggaggg gcgggggcgc
     2281 aaccgcggcg accagctcac cgggttctgc cgtggggagg gagcagaggc caggatgcac
     2341 gcgtccttct gaaggaacag ggtctcggtc tccggaaagg agaaagaatc tagagttcat
     2401 agcggagcag gggtcgcgga gggggctcga gctgtagcgc tggggggccg tgatgcccat
     2461 ttctagattt tggatacccg ctgggacgtg gtaagtgcgc gcctgggact gccgagaagg
     2521 agctcccgct ttcgcactcg aatccgggga gccggcgcgg agaggcggcc cctcaaagct
     2581 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2641 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2701 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2761 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2821 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2881 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2941 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3001 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3061 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3121 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3181 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3241 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3301 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3361 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3421 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3481 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3541 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3601 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3661 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3721 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3781 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3841 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3901 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3961 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4021 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4081 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4141 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4201 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4261 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4321 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4381 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4441 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4501 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4561 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4621 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4681 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4741 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4801 cgaggccctt tcgtc
//
