LOCUS       sequence-416196-        5247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Blast-2A-ultraID 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 5247)
  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 5247)
  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-ultraID-LMNB1
FEATURES             Location/Qualifiers
     source          1..5247
                     /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"
     primer_bind     complement(3026..3042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3026..3042)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3039..3061)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3050..3066
                     /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(3074..3080,3081..3098,3099..3104))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3119..3140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3257..3274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3428..4016)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3508..3527)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4187..4978,4979..5047))
                     /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     4810..4829
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5048..5152)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5220..5238
                     /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 ggatcaatgt tcaagaacct gatctggctg aaggaggtgg acagcaccca ggagagactg
     1801 aaggagtgga acgtgtccta cggcaccgcc ctggtggccg acagacagac caagggcaga
     1861 ggcggccccg gcagaaagtg gctgagccag gagggcggcc tgtacttcag cttcctgctg
     1921 aaccccaagg agttcgagaa cctgctgcag ctgcccctgg tgctgggcct gagcgtgagc
     1981 gaggccctgg aggagatcac cgagatcccc ttcagcctga agtggcccaa cgacgtgtac
     2041 ttccaggaga agaaggtgag cggcgtgctg tgcgagctga gcaaggacaa gctgatcgtg
     2101 ggcatcggca tcaacgtgaa ccagagagag atccccgagg agatcaagga cagagccacc
     2161 accctgtacg agatcaccgg caaggactgg gacagaaagg aggtgctgct gaaggtgctg
     2221 aagagaatca gcgagaacct gaagaagttc aaggagaagg gtggaggcgg atctctcgag
     2281 ggaggaagcg gaggatctgg cggtaccgcg actgcgaccc ccgtgccgcc gcggatgggc
     2341 agccgcgctg gcggccccac cacgccgctg agccccacgc gcctgtcgcg gctccaggag
     2401 aaggaggagc tgcgcgagct caatgaccgg ctggcggtgt acatcgacaa ggtgcgcagc
     2461 ctggagacgg agaacagcgc gctgcagctg caggtgacgg agcgcgagga ggtgcgcggc
     2521 cgtgagctca ccggcctcaa ggcgctctac gagaccgagc tggccgacgc gcgacgcgcg
     2581 ctcgacgaca cggcccgcga gcgcgccaag ctgcagatcg agctgggcaa gtgcaaggcg
     2641 gaacacgacc agctgctcct caagtgagtg ctagctggcg gccgcgttag cgccaaggag
     2701 gggcgggggc gcaaccgcgg cgaccagctc accgggttct gccgtgggga gggagcagag
     2761 gccaggatgc acgcgtcctt ctgaaggaac agggtctcgg tctccggaaa ggagaaagaa
     2821 tctagagttc atagcggagc aggggtcgcg gagggggctc gagctgtagc gctggggggc
     2881 cgtgatgccc atttctagat tttggatacc cgctgggacg tggtaagtgc gcgcctggga
     2941 ctgccgagaa ggagctcccg ctttcgcact cgaatccggg gagccggcgc ggagaggcgg
     3001 cccctcaaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3061 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3121 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3181 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3241 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3301 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3361 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3421 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3481 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3541 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3601 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3661 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3721 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3781 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3841 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3901 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3961 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4021 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4081 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4141 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4201 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4261 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4321 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4381 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4441 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4501 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4561 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4621 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4681 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4741 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4801 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4861 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4921 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4981 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5041 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5101 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5161 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     5221 ataggcgtat cacgaggccc tttcgtc
//
