LOCUS       sequence-413796-        5116 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mNeon-2A-Blast insertion into the C-terminus of 
            the TUBB4B locus for tubulin visualization. To be co-transfected 
            with sgRNA plasmid px330-PITCh-TUBB4B Addgene #207785.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5116)
  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 5116)
  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-mNeon-Blast-TUBB4B
FEATURES             Location/Qualifiers
     source          1..5116
                     /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             978..1685
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1233..1242
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1695..1721
                     /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             1695..1721
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1725..1751
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1758..1791)
                     /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             1803..1859
                     /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             1866..2261
                     /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(2268..2301)
                     /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(2895..2911)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2895..2911)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2908..2930)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2919..2935
                     /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(2943..2949,2950..2967,2968..2973))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2988..3009
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3126..3143)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3297..3885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3377..3396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4056..4847,4848..4916))
                     /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     4679..4698
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4917..5021)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5089..5107
                     /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 cctgcacttc ttcatgcccg
      421 gctttgcccc actgaccagc cggggcagcc agcagtaccg ggcgctgacc gtgcccgagc
      481 tcacccagca gatgtttgat gccaagaaca tgatggctgc ctgcgacccc cgccatggcc
      541 gctacctgac ggttgccgcc gtgttcaggg gccgcatgtc catgaaggag gtggatgagc
      601 aaatgcttaa tgtccaaaac aaaaacagca gctattttgt tgagtggatc cccaacaatg
      661 tgaaaacggc tgtctgtgac atcccacctc gggggctaaa aatgtccgcc accttcattg
      721 gcaacagcac ggccatccag gagctgttca agcgcatctc cgagcagttc acggccatgt
      781 tccggcgcaa ggccttcctg cactggtaca cgggcgaggg catggacgag atggagttca
      841 ccgaggccga gagcaacatg aatgacctgg tgtccgagta ccagcagtac caggatgcca
      901 cagccgagga ggagggcgag ttcgaggagg aggctgagga ggaggtggcc ggaggaagcg
      961 gaggatctgg cggtaccatg gtgagcaagg gcgaggagga taacatggcc tctctcccag
     1021 cgacacatga gttacacatc tttggctcca tcaacggtgt ggactttgac atggtgggtc
     1081 agggcaccgg caatccaaat gatggttatg aggagttaaa cctgaagtcc accaagggtg
     1141 acctccagtt ctccccctgg attctggtcc ctcatatcgg gtatggcttc catcagtacc
     1201 tgccctaccc tgacgggatg tcgcctttcc aggccgccat ggtagatggc tccggatacc
     1261 aagtccatcg cacaatgcag tttgaagatg gtgcctccct tactgttaac taccgctaca
     1321 cctacgaggg aagccacatc aaaggagagg cccaggtgaa ggggactggt ttccctgctg
     1381 acggtcctgt gatgaccaac tcgctgaccg ctgcggactg gtgcaggtcg aagaagactt
     1441 accccaacga caaaaccatc atcagtacct ttaagtggag ttacaccact ggaaatggca
     1501 agcgctaccg gagcactgcg cggaccacct acacctttgc caagccaatg gcggctaact
     1561 atctgaagaa ccagccgatg tacgtgttcc gtaagacgga gctcaagcac tccaagaccg
     1621 agctcaactt caaggagtgg caaaaggcct ttaccgatgt gatgggcatg gacgagctgt
     1681 acaagggctc tggctacccc tacgacgtgc ccgactacgc cggctatccg tatgatgtcc
     1741 cggactatgc aacgcgttac cgttcgtata gcatacatta tacgaagtta tctggatccg
     1801 gagctactaa cttcagcctg ctgaagcagg ccggagacgt ggaggagaac cctggacctt
     1861 ctagaatggc caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
     1921 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg
     1981 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
     2041 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
     2101 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
     2161 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
     2221 ggattcgtga attgctgccc tctggttatg tgtgggaggg ccccgggata acttcgtata
     2281 gcatacatta tacgaacggt agcctcgaga ccggtaagag caagtgatag agacttcagt
     2341 cactggtgaa agcagggaag cagtgtgaac tctttattca ctcccagcct gtcctgtggc
     2401 ctgtcccact gtgtgcactt gctgttttcc ctgtccacat ccatgctgta cagacaccac
     2461 cattaaagca ttttcatagt gtgtggtttc gctttgccca ttccttccag taggccctcc
     2521 gggtgctgtt gccgaatgtc agggcgtagt tgtcctgcat ggctgcgggg agccaggctg
     2581 cagctgagct gggcttatgt gcccaggacc agcatgggct ggacacccag gcttcagaga
     2641 agtgtggggc tgccctgggc cctgggtatc tgcttggtgg ggaacttgct tgttgaaggc
     2701 aggcctgtcc tgttccaggt acccaacctt ggggctcggc ctgtgtctgg gacttaggtg
     2761 ggtccagtga ccccccttcc aaaaggggta gggaggatgg cagacaatcc taccccaacc
     2821 aaggtgggac cctggtaatg gtcagacagg tggtcctgaa ccagcaatga agggtgaagc
     2881 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     2941 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3001 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3061 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3121 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3181 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3241 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3301 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3361 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3421 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3481 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3541 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3601 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3661 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3721 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3781 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3841 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3901 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3961 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4021 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4081 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4141 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4201 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4261 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4321 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4381 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4441 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4501 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4561 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4621 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4681 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4741 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4801 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4861 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4921 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4981 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5041 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     5101 acgaggccct ttcgtc
//
