LOCUS       sequence-413797-        5631 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Puro-2A-moxGFP 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 5631)
  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 5631)
  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-moxGFP-LMNB1
FEATURES             Location/Qualifiers
     source          1..5631
                     /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             1876..1902
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1876..1902
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1906..1932
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1942..1944,1945..1947,1948..2658)
                     /codon_start=1
                     /product="monomeric GFP variant optimized for use in 
                     oxidizing environments such as the secretory pathway 
                     (Costantini et al., 2015)"
                     /label=moxGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1987..2008)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2595..2616
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3410..3426)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3410..3426)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3423..3445)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3434..3450
                     /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(3458..3464,3465..3482,3483..3488))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3503..3524
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3641..3658)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3812..4400)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3892..3911)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4571..5362,5363..5431))
                     /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     5194..5213
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5432..5536)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5604..5622
                     /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 gtcaccctcg 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 cgcgttaccc ctacgacgtg cccgactacg ccggctatcc gtatgatgtc
     1921 ccggactatg caggaagcgg aatggtgagc aagggcgagg agctgttcac cggggtggtg
     1981 cccatcctgg tcgagctgga cggcgacgtg aacggccaca agttctccgt gcggggcgag
     2041 ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatcagcac caccggcaag
     2101 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gagcttctcc
     2161 cgctaccccg accacatgaa gcgccacgac ttcttcaaga gcgccatgcc cgaaggctac
     2221 gtccaggagc gcaccatctc cttcaaggac gacggcacct acaagacccg cgccgaggtg
     2281 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2341 gacggcaaca tcctggggca caagctggag tacaacttca actcccacaa cgtctatatc
     2401 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca taacgtggag
     2461 gacggctccg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     2521 gtgctgctgc ccgacaacca ctacctgtcc acccagtcca agctgtccaa agaccccaac
     2581 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactcacggc
     2641 atggacgagc tgtacaagct cgagggagga agcggaggat ctggcggtac cgcgactgcg
     2701 acccccgtgc cgccgcggat gggcagccgc gctggcggcc ccaccacgcc gctgagcccc
     2761 acgcgcctgt cgcggctcca ggagaaggag gagctgcgcg agctcaatga ccggctggcg
     2821 gtgtacatcg acaaggtgcg cagcctggag acggagaaca gcgcgctgca gctgcaggtg
     2881 acggagcgcg aggaggtgcg cggccgtgag ctcaccggcc tcaaggcgct ctacgagacc
     2941 gagctggccg acgcgcgacg cgcgctcgac gacacggccc gcgagcgcgc caagctgcag
     3001 atcgagctgg gcaagtgcaa ggcggaacac gaccagctgc tcctcaagtg agtgctagct
     3061 ggcggccgcg ttagcgccaa ggaggggcgg gggcgcaacc gcggcgacca gctcaccggg
     3121 ttctgccgtg gggagggagc agaggccagg atgcacgcgt ccttctgaag gaacagggtc
     3181 tcggtctccg gaaaggagaa agaatctaga gttcatagcg gagcaggggt cgcggagggg
     3241 gctcgagctg tagcgctggg gggccgtgat gcccatttct agattttgga tacccgctgg
     3301 gacgtggtaa gtgcgcgcct gggactgccg agaaggagct cccgctttcg cactcgaatc
     3361 cggggagccg gcgcggagag gcggcccctc aaagcttggc gtaatcatgg tcatagctgt
     3421 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3481 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3541 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3601 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3661 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3721 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3781 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3841 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3901 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3961 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4021 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4081 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4141 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4201 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4261 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4321 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4381 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4441 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4501 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4561 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4621 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4681 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4741 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4801 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4861 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4921 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4981 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5041 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5101 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5161 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5221 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5281 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5341 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5401 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5461 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5521 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     5581 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
