LOCUS       sequence-416197-        5511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Blast-2A-miniTurbo 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 5511)
  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 5511)
  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-miniTurbo-LMNB1
FEATURES             Location/Qualifiers
     source          1..5511
                     /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             1753..2523
                     /codon_start=1
                     /product="N-terminally truncated mutant of the E. coli 
                     biotin ligase BioID engineered for improved catalytic 
                     efficiency (Branon et al., 2018)"
                     /label=miniTurbo
                     /translation="IPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACI
                     AEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADK
                     VRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQ
                     EAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIF
                     GISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     primer_bind     complement(3290..3306)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3290..3306)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3303..3325)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3314..3330
                     /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(3338..3344,3345..3362,3363..3368))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3383..3404
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3521..3538)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3692..4280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3772..3791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4451..5242,5243..5311))
                     /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"
     promoter        complement(5312..5416)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5484..5502
                     /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 ggatcaatgg cgatcccgct gctgaacgct aaacagattc tgggacagct ggacggcggg
     1801 agcgtggcag tcctgcctgt ggtcgactcc accaatcagt acctgctgga tcgaatcggc
     1861 gagctgaaga gtggggatgc ttgcattgca gaatatcagc aggcagggag aggaagcaga
     1921 gggaggaaat ggttctctcc ttttggagct aacctgtacc tgagtatgtt ttggcgcctg
     1981 aagcggggac cagcagcaat cggcctgggc ccggtcatcg gaattgtcat ggcagaagcg
     2041 ctgcgaaagc tgggagcaga caaggtgcga gtcaaatggc ccaatgacct gtatctgcag
     2101 gatagaaagc tggcaggcat cctggtggag ctggccggaa taacaggcga tgctgcacag
     2161 atcgtcattg gcgccgggat taacgtggct atgaggcgcg tggaggaaag cgtggtcaat
     2221 cagggctgga tcacactgca ggaagcaggg attaacctgg acaggaatac tctggccgct
     2281 atgctgatcc gagagctgcg ggcagccctg gaactgttcg agcaggaagg cctggctcca
     2341 tatctgtcac ggtgggagaa gctggataac ttcatcaata gacccgtgaa gctgatcatt
     2401 ggggacaaag agattttcgg gattagccgg gggattgata aacagggagc cctgctgctg
     2461 gaacaggacg gagttatcaa accctggatg ggcggagaaa tcagtctgcg gtctgccgaa
     2521 aagggtggag gcggatctct cgagggagga agcggaggat ctggcggtac cgcgactgcg
     2581 acccccgtgc cgccgcggat gggcagccgc gctggcggcc ccaccacgcc gctgagcccc
     2641 acgcgcctgt cgcggctcca ggagaaggag gagctgcgcg agctcaatga ccggctggcg
     2701 gtgtacatcg acaaggtgcg cagcctggag acggagaaca gcgcgctgca gctgcaggtg
     2761 acggagcgcg aggaggtgcg cggccgtgag ctcaccggcc tcaaggcgct ctacgagacc
     2821 gagctggccg acgcgcgacg cgcgctcgac gacacggccc gcgagcgcgc caagctgcag
     2881 atcgagctgg gcaagtgcaa ggcggaacac gaccagctgc tcctcaagtg agtgctagct
     2941 ggcggccgcg ttagcgccaa ggaggggcgg gggcgcaacc gcggcgacca gctcaccggg
     3001 ttctgccgtg gggagggagc agaggccagg atgcacgcgt ccttctgaag gaacagggtc
     3061 tcggtctccg gaaaggagaa agaatctaga gttcatagcg gagcaggggt cgcggagggg
     3121 gctcgagctg tagcgctggg gggccgtgat gcccatttct agattttgga tacccgctgg
     3181 gacgtggtaa gtgcgcgcct gggactgccg agaaggagct cccgctttcg cactcgaatc
     3241 cggggagccg gcgcggagag gcggcccctc aaagcttggc gtaatcatgg tcatagctgt
     3301 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     3361 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3421 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3481 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3541 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3601 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3661 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3721 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3781 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3841 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3901 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3961 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4021 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4081 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4141 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4201 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4261 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4321 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4381 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4441 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4501 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4561 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4621 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4681 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4741 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4801 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4861 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4921 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4981 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5041 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5101 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5161 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5221 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5281 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     5341 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     5401 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     5461 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
