LOCUS       sequence-416184-        4870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for miRFP670nano3-2A-Blast insertion into the 
            C-terminus of the H3C2 locus for nuclei visualization. To be 
            co-transfected with sgRNA plasmid px330-PITCh-H3C2 Addgene #207780.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4870)
  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 4870)
  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-C-miRFP670nano3-Blast-H3C2
FEATURES             Location/Qualifiers
     source          1..4870
                     /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"
     CDS             1449..1490
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1512..1545)
                     /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             1557..1613
                     /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"
     CDS             1620..2015
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     protein_bind    complement(2022..2055)
                     /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)."
     primer_bind     complement(2649..2665)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2649..2665)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2662..2684)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2673..2689
                     /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(2697..2703,2704..2721,2722..2727))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2742..2763
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2880..2897)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3051..3639)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3131..3150)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3810..4601,4602..4670))
                     /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     4433..4452
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4671..4775)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4843..4861
                     /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 cgatggacca atccaagaag
      421 ggcgcgggga tttttgaatt ttcttgggtc caatagttgg tggtctgact ctataaaaga
      481 agagtagctc tttcctttcc tccacagacg tctctgcagg caagcttttc tgtggttttg
      541 ccatggctcg tactaaacag acagctcgga aatccaccgg cggtaaagcg ccacgcaagc
      601 agctggctac caaggctgct cgcaagagcg cgccggctac cggcggcgtg aaaaagcctc
      661 accgttaccg cccgggcact gtggctctgc gcgagatccg ccgctaccaa aagtcgaccg
      721 agttgctgat tcggaagctg ccgttccagc gcctggtgcg agaaatcgcc caagacttca
      781 agaccgatct tcgcttccag agctctgcgg tgatggcgct gcaggaggct tgtgaggcct
      841 acttggtagg gctctttgag gacacaaacc tttgcgccat ccatgctaag cgagtgacta
      901 ttatgcccaa agacatccag ctcgctcggc gcattcgcgg agaaagagcg ggaggaagcg
      961 gaggatctgg cggtaccggt ggaggcggat ctatggcaaa cctggacaag atgctgaaca
     1021 ccaccgtgac cgaggtgcgc aagttcctgc aagcagacag agtgtgcgtg ttcaagttcg
     1081 aggaagatta ctccggcacc gtcagccacg aagccgtgga cgacagatgg attagcatcc
     1141 tgaaaaccca ggtgcaggac agatacttca tggaaaccag aggcgaggaa tacgtccacg
     1201 gcagatacca ggccatcgcc gacatctaca cagccaatct ggtcgagtgc tacagagacc
     1261 tgctgatcga gtttcaggtg cgggccattc tggctgtccc catcctgcaa ggcaagaagc
     1321 tgtggggcct gctggtggcc caccaactgg ccggccctcg ggagtggcag acctgggaaa
     1381 tcgacttcct gaaacagcaa gccgtggtga tgggcatcgc catccagcag agcggaggtg
     1441 gcggttccgg caagcccatc cccaaccccc tgctgggcct ggacagcacc ggaggtggcg
     1501 gatcaacgcg ttaccgttcg tatagcatac attatacgaa gttatctgga tccggagcta
     1561 ctaacttcag cctgctgaag caggccggag acgtggagga gaaccctgga ccttctagaa
     1621 tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca
     1681 acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc
     1741 gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg
     1801 tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa
     1861 atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc
     1921 atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc
     1981 gtgaattgct gccctctggt tatgtgtggg agggccccgg gataacttcg tatagcatac
     2041 attatacgaa cggtagcctc gagaccggta agagcaagtg ataaatgtaa agtcactttt
     2101 tcatcagtct taaaacccaa agtctctttt cagagccacc cacttattcc aacgaaagta
     2161 gctgtgataa ttttttgttg tcttaacaga acaaatttct aaggaccccc ccggaaagca
     2221 ttagactatg gtcttaaagt tgattaacag aaataacggt ttggtcagtc ttgcagtgta
     2281 ggttatttct gaccttatta aggtgctatt tggagagaag ctgtgtaagt ccactatcat
     2341 tcaggcctct agcttgctat gattagcatt tgtttaaaca actttgtaag agtaagggaa
     2401 aaatctggta agtagttaac tggcgcttac taggcatttt tgcaaagctt tgaaaagatt
     2461 agaaaattgt gtcttgcgag ttccagtgtc ttcctcaaaa tgcttaggaa gattttctca
     2521 gctcaataca tagtccccta ggttttctca tatattatat ctatatctac atatatatct
     2581 actgttaaat tcatttggct gttaacatta acctgaaatt tattctggtg aagcttggcg
     2641 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     2701 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     2761 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2821 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2881 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2941 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3001 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3061 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3121 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3181 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3241 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     3301 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3361 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3421 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3481 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3541 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3601 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3661 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3721 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3781 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3841 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3901 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3961 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4021 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4081 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4141 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4201 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4261 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     4321 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4381 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4441 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4501 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4561 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4621 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4681 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4741 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4801 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     4861 ccctttcgtc
//
