LOCUS       sequence-415638-        5128 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Blast-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 5128)
  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 5128)
  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-moxGFP-LMNB1
FEATURES             Location/Qualifiers
     source          1..5128
                     /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(972..1005)
                     /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             1014..1409
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1419..1475
                     /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(1482..1515)
                     /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             1524..1550
                     /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             1524..1550
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1554..1580
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1590..1592,1593..1595,1596..2306)
                     /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(1635..1656)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2243..2264
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2907..2923)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2907..2923)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2920..2942)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2931..2947
                     /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(2955..2961,2962..2979,2980..2985))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3000..3021
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3138..3155)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3309..3897)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3389..3408)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4068..4859,4860..4928))
                     /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     4691..4710
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4929..5033)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5101..5119
                     /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 cgcccccgaa ccgccgaagc
      421 gcgctgcctg ctccccattg gcccatggta gtcacgtgga ggcgccgggg cgtgccggcc
      481 atgttgggga gtgcggcgcc gcggcccgcg ccacctccgc cccccgcggc ttgcctccag
      541 cccgcccctc ccggccctcc tccccccgcc cgccgctccg tgcagcctga gaggaaacaa
      601 agtgctgcga gcaggagacg gcggcggcgc gaaccctgct gggcctccag tcaccctcgt
      661 cttgcatttt cccgcgtgcg tgtgtgagtg ggtgtgtgtg ttttcttaca aagggtattt
      721 cgcgatcgat cgattgattc gtagttcccc cccgcgcgcc tttgcccttt gtgctgtaat
      781 cgagctcccg ccatcccagg tgcttctccg ttcctctaaa cgccagcgtc tggacgtgag
      841 cgcaggtcgc cggtttgtgc cttcggtccc cgcttcgccc cctgccgtcc cctccttatc
      901 acggtcccgc tcgcggcctc gccgccccgc tgtctccgcc gcccgccatg ggcaagagca
      961 agaccggtag ctaccgttcg tatagcatac attatacgaa gttatctgct agcatggcca
     1021 agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca
     1081 tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct
     1141 tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg
     1201 gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga
     1261 acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg
     1321 ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat
     1381 tgctgccctc tggttatgtg tgggagggcg gatccggagc tactaacttc agcctgctga
     1441 agcaggccgg agacgtggag gagaaccctg gaccttctag aataacttcg tatagcatac
     1501 attatacgaa cggtagcacg cgttacccct acgacgtgcc cgactacgcc ggctatccgt
     1561 atgatgtccc ggactatgca ggaagcggaa tggtgagcaa gggcgaggag ctgttcaccg
     1621 gggtggtgcc catcctggtc gagctggacg gcgacgtgaa cggccacaag ttctccgtgc
     1681 ggggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc atcagcacca
     1741 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcaga
     1801 gcttctcccg ctaccccgac cacatgaagc gccacgactt cttcaagagc gccatgcccg
     1861 aaggctacgt ccaggagcgc accatctcct tcaaggacga cggcacctac aagacccgcg
     1921 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1981 tcaaggagga cggcaacatc ctggggcaca agctggagta caacttcaac tcccacaacg
     2041 tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccata
     2101 acgtggagga cggctccgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2161 acggccccgt gctgctgccc gacaaccact acctgtccac ccagtccaag ctgtccaaag
     2221 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2281 ctcacggcat ggacgagctg tacaagctcg agggaggaag cggaggatct ggcggtaccg
     2341 cgactgcgac ccccgtgccg ccgcggatgg gcagccgcgc tggcggcccc accacgccgc
     2401 tgagccccac gcgcctgtcg cggctccagg agaaggagga gctgcgcgag ctcaatgacc
     2461 ggctggcggt gtacatcgac aaggtgcgca gcctggagac ggagaacagc gcgctgcagc
     2521 tgcaggtgac ggagcgcgag gaggtgcgcg gccgtgagct caccggcctc aaggcgctct
     2581 acgagaccga gctggccgac gcgcgacgcg cgctcgacga cacggcccgc gagcgcgcca
     2641 agctgcagat cgagctgggc aagtgcaagg cggaacacga ccagctgctc ctcaagtgag
     2701 tgctagctgg cggccgcgtt agcgccaagg aggggcgggg gcgcaaccgc ggcgaccagc
     2761 tcaccgggtt ctgccgtggg gagggagcag aggccaggat gcacgcgtcc ttctgaagga
     2821 acagggtctc ggtctccgga aaggagaaag aatctagagt tcatagcgga gcaggggtcg
     2881 cggaggggaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2941 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3001 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3061 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3121 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3181 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3241 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3301 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3361 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3421 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3481 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3541 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3601 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3661 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3721 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3781 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3841 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3901 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3961 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4021 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4081 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4141 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4201 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4261 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4321 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4381 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4441 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4501 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4561 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4621 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4681 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4741 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4801 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4861 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4921 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4981 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5041 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     5101 aataggcgta tcacgaggcc ctttcgtc
//
