LOCUS       sequence-451729-        5643 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Puro-2A-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 5643)
  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 5643)
  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-Puro-mStayGold-LMNB1
FEATURES             Location/Qualifiers
     source          1..5643
                     /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"
     protein_bind    complement(1123..1156)
                     /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)."
     CDS             1165..1761
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1165..1184)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1661..1681
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1771..1827
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    complement(1834..1867)
                     /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)."
     CDS             1891..1932
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1948..2625
                     /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(3422..3438)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3422..3438)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3435..3457)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3446..3462
                     /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(3470..3476,3477..3494,3495..3500))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3515..3536
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3653..3670)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3824..4412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3904..3923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4583..5374,5375..5443))
                     /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     5206..5225
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5444..5548)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5616..5634
                     /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 gctaccgttc gtatagcata
     1141 cattatacga agttatctgc tagcatgacc gagtacaagc ccacggtgcg cctcgccacc
     1201 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     1261 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     1321 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     1381 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     1441 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     1501 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     1561 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     1621 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     1681 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     1741 atgacccgca agcccggtgc cggatccgga gctactaact tcagcctgct gaagcaggcc
     1801 ggagacgtgg aggagaaccc tggaccttct agaataactt cgtatagcat acattatacg
     1861 aacggtagca cgcgtggagg tggcggttcc ggcaagccca tccccaaccc cctgctgggc
     1921 ctggacagca ccggaggtgg cggaagcatg gtgtctacag gcgaggaact gtttaccggc
     1981 gtggtgccct tcaagttcca gctgaagggc accatcaacg gcaagagctt caccgtggaa
     2041 ggcgagggcg agggcaatag ccacgagggc agccacaaag gcaagtatgt gtgcaccagc
     2101 ggcaaactgc caatgtcttg ggccgccctg ggaactagct tcggctatgg catgaaatac
     2161 tataccaagt accccagcgg cctgaaaaac tggttccacg aggtgatgcc tgagggcttc
     2221 acctacgaca gacacatcca gtacaagggc gacggcagca tccacgccaa gcaccagcac
     2281 ttcatgaaga acggcaccta ccacaacatc gtggagttca ccggccagga cttcaaggag
     2341 aacagccccg tgctgaccgg cgacatggac gtgagcctgc ccaacgaggt gcagcacatc
     2401 cccattgatg acggcgtgga gtgcacagtg accctgcagt accctctgct gagcgacgaa
     2461 agcaagtgcg tggaagccta tcagaacacc atcatcaagc ccctgcacaa tcagccagcc
     2521 cccgatgtgc catttcactg gatcagaaag cagtacaccc agagcaagga cgacaccgag
     2581 gagagagacc acatcatcca gagcgagacc ctggaggccc acctgccttg gcatgagcct
     2641 agcgcaagcg cagtgggagg tggcggttcc ctcgagggag gaagcggagg atctggcggt
     2701 accgcgactg cgacccccgt gccgccgcgg atgggcagcc gcgctggcgg ccccaccacg
     2761 ccgctgagcc ccacgcgcct gtcgcggctc caggagaagg aggagctgcg cgagctcaat
     2821 gaccggctgg cggtgtacat cgacaaggtg cgcagcctgg agacggagaa cagcgcgctg
     2881 cagctgcagg tgacggagcg cgaggaggtg cgcggccgtg agctcaccgg cctcaaggcg
     2941 ctctacgaga ccgagctggc cgacgcgcga cgcgcgctcg acgacacggc ccgcgagcgc
     3001 gccaagctgc agatcgagct gggcaagtgc aaggcggaac acgaccagct gctcctcaag
     3061 tgagtgctag ctggcggccg cgttagcgcc aaggaggggc gggggcgcaa ccgcggcgac
     3121 cagctcaccg ggttctgccg tggggaggga gcagaggcca ggatgcacgc gtccttctga
     3181 aggaacaggg tctcggtctc cggaaaggag aaagaatcta gagttcatag cggagcaggg
     3241 gtcgcggagg gggctcgagc tgtagcgctg gggggccgtg atgcccattt ctagattttg
     3301 gatacccgct gggacgtggt aagtgcgcgc ctgggactgc cgagaaggag ctcccgcttt
     3361 cgcactcgaa tccggggagc cggcgcggag aggcggcccc tcaaagcttg gcgtaatcat
     3421 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3481 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3541 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3601 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3661 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3721 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3781 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3841 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3901 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3961 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4021 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4081 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4141 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4201 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4261 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4321 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4381 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4441 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4501 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4561 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4621 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4681 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4741 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4801 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4861 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4921 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4981 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5041 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5101 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5161 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5221 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5281 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5341 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5401 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5461 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5521 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     5581 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     5641 gtc
//
