LOCUS       sequence-416195-        5055 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Blast-2A-dTAG insertion into the N-terminus of 
            the LMNB1 locus. 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 5055)
  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 5055)
  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-Blast-dTAG-LMNB1
FEATURES             Location/Qualifiers
     source          1..5055
                     /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..1560
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1570..1626
                     /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(1633..1666)
                     /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             1690..1731
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1747..2067
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     primer_bind     complement(2834..2850)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2834..2850)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2847..2869)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2858..2874
                     /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(2882..2888,2889..2906,2907..2912))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2927..2948
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3065..3082)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3236..3824)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3316..3335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3995..4786,4787..4855))
                     /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     4618..4637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4856..4960)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5028..5046
                     /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 tagcatggcc aagcctttgt ctcaagaaga atccaccctc
     1201 attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc
     1261 agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact
     1321 gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac
     1381 ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg
     1441 tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat
     1501 ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc
     1561 ggatccggag ctactaactt cagcctgctg aagcaggccg gagacgtgga ggagaaccct
     1621 ggaccttcta gaataacttc gtatagcata cattatacga acggtagcac gcgtggaggt
     1681 ggcggttccg gcaagcccat ccccaacccc ctgctgggcc tggacagcac cggaggtggc
     1741 ggatcaggag tgcaggtgga aaccatctcc ccaggagacg ggcgcacctt ccccaagcgc
     1801 ggccagacct gcgtggtgca ctacaccggg atgcttgaag atggaaagaa agttgattcc
     1861 agccgggaca gaaacaagcc ctttaagttt atgctaggca agcaggaggt gatccgaggc
     1921 tgggaagaag gggttgccca gatgagtgtg ggtcagagag ccaaactgac tatatctcca
     1981 gattatgcct atggtgccac tgggcaccca ggcatcatcc caccacatgc cactctcgtc
     2041 ttcgatgtgg agcttctaaa actggaaggt ggaggcggat ctctcgaggg aggaagcgga
     2101 ggatctggcg gtaccgcgac tgcgaccccc gtgccgccgc ggatgggcag ccgcgctggc
     2161 ggccccacca cgccgctgag ccccacgcgc ctgtcgcggc tccaggagaa ggaggagctg
     2221 cgcgagctca atgaccggct ggcggtgtac atcgacaagg tgcgcagcct ggagacggag
     2281 aacagcgcgc tgcagctgca ggtgacggag cgcgaggagg tgcgcggccg tgagctcacc
     2341 ggcctcaagg cgctctacga gaccgagctg gccgacgcgc gacgcgcgct cgacgacacg
     2401 gcccgcgagc gcgccaagct gcagatcgag ctgggcaagt gcaaggcgga acacgaccag
     2461 ctgctcctca agtgagtgct agctggcggc cgcgttagcg ccaaggaggg gcgggggcgc
     2521 aaccgcggcg accagctcac cgggttctgc cgtggggagg gagcagaggc caggatgcac
     2581 gcgtccttct gaaggaacag ggtctcggtc tccggaaagg agaaagaatc tagagttcat
     2641 agcggagcag gggtcgcgga gggggctcga gctgtagcgc tggggggccg tgatgcccat
     2701 ttctagattt tggatacccg ctgggacgtg gtaagtgcgc gcctgggact gccgagaagg
     2761 agctcccgct ttcgcactcg aatccgggga gccggcgcgg agaggcggcc cctcaaagct
     2821 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2881 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2941 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3001 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3061 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3121 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3181 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3241 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3301 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3361 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3421 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3481 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3541 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3601 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3661 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3721 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3781 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3841 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3901 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3961 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4021 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4081 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4141 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4201 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4261 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4321 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4381 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4441 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4501 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4561 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4621 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4681 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4741 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4801 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4861 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4921 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4981 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     5041 cgaggccctt tcgtc
//
