LOCUS       sequence-415639-        5419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mNeon-2A-Puro insertion into the C-terminus of 
            the TOMM20 locus. For mitochondria visualization. To be 
            co-transfected with sgRNA plasmid px330-PITCh-TOMM20 (Addgene 
            #207789).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5419)
  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 5419)
  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-Puro-TOMM20
FEATURES             Location/Qualifiers
     source          1..5419
                     /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             1029..1736
                     /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      1284..1293
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1746..1772
                     /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             1746..1772
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1776..1802
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(1809..1842)
                     /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             1854..1910
                     /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             1917..2513
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1917..1936)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2413..2433
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2520..2553)
                     /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(3198..3214)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3198..3214)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3211..3233)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3222..3238
                     /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(3246..3252,3253..3270,3271..3276))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3291..3312
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3429..3446)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3600..4188)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3680..3699)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4359..5150,5151..5219))
                     /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     4982..5001
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5220..5324)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5392..5410
                     /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 ctggttttag ttacataatt
      421 gactgatgta atattatgta atgatggctg tacacagtgt ctcatgccct ataatcctag
      481 cactcatttg agctcaggag ttcaaggcca gcctgggcaa catggtaaaa ccctgtctcc
      541 accaaaaata aaaaaaaaat tagccgggca tggtggcatg agtctgtggt tccagctact
      601 caggaggctg aggtggggga ggatcgcttg agcccaggag tcagaggttg cagtgagctg
      661 agattgtgct actgcactcc agcctgggtt acagagaccc catctgaatt aaaaacatat
      721 ataatgtaat gatctgcctc ctttgttaac ttgacttttg aaatgggatt gtcagtagta
      781 tgatcattgt tttcttggat gccgactgtg tgtaaagtgt tacattttga attaaatgtc
      841 agaatgggtg aactttacta agattcaatt ctttgaatac aaagagcatt ttattttgaa
      901 gttagaatcc taattaaatg cttatgacac tttaaaaaat tatttttttt ttctttcaga
      961 gaattgtaag tgctcagagc ttagctgaag atgatgttga aggaggaagc ggaggatctg
     1021 gcggtaccat ggtgagcaag ggcgaggagg ataacatggc ctctctccca gcgacacatg
     1081 agttacacat ctttggctcc atcaacggtg tggactttga catggtgggt cagggcaccg
     1141 gcaatccaaa tgatggttat gaggagttaa acctgaagtc caccaagggt gacctccagt
     1201 tctccccctg gattctggtc cctcatatcg ggtatggctt ccatcagtac ctgccctacc
     1261 ctgacgggat gtcgcctttc caggccgcca tggtagatgg ctccggatac caagtccatc
     1321 gcacaatgca gtttgaagat ggtgcctccc ttactgttaa ctaccgctac acctacgagg
     1381 gaagccacat caaaggagag gcccaggtga aggggactgg tttccctgct gacggtcctg
     1441 tgatgaccaa ctcgctgacc gctgcggact ggtgcaggtc gaagaagact taccccaacg
     1501 acaaaaccat catcagtacc tttaagtgga gttacaccac tggaaatggc aagcgctacc
     1561 ggagcactgc gcggaccacc tacacctttg ccaagccaat ggcggctaac tatctgaaga
     1621 accagccgat gtacgtgttc cgtaagacgg agctcaagca ctccaagacc gagctcaact
     1681 tcaaggagtg gcaaaaggcc tttaccgatg tgatgggcat ggacgagctg tacaagggct
     1741 ctggctaccc ctacgacgtg cccgactacg ccggctatcc gtatgatgtc ccggactatg
     1801 caacgcgtta ccgttcgtat agcatacatt atacgaagtt atctggatcc ggagctacta
     1861 acttcagcct gctgaagcag gccggagacg tggaggagaa ccctggacct tctagaatga
     1921 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     1981 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     2041 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     2101 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     2161 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     2221 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     2281 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     2341 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     2401 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     2461 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcccccggga
     2521 taacttcgta tagcatacat tatacgaacg gtagcctcga gaccggtaag agcaagtgat
     2581 gagaaacaaa tgtcaacata ataaaatctc agttaaaaat attttaaaaa ttcttggtag
     2641 ttgagcagct ctgggggaat aagggcaaat atgcttgtta tgaactacac tgaaatctac
     2701 caaagttaat gtttactttg tgtagatcca tttgtctatt ttatttattt ttcccagtga
     2761 aaagtgtatt ttgatagaga acttttcatt ctataaatac actatgagtt actaaaatat
     2821 catggatttt gtttattcct gaaacatagt tacatagtta aactgtacat atgacatggc
     2881 ttatgttaaa aatacccagt gctcagtttt gaaagatagg caaaaaaaaa aaagtatagg
     2941 agaaactgaa gaatgtacac ttttttagag ggcacatttt gctgtaaatc tggaaatttg
     3001 atagacttga ctgtgtttgt gaaaactgag cattaaaggt tttgattgat cctttctttc
     3061 catttaatct ctgagacgta aatatgtgag gtgtgctgct gtgctgggtt aacagcttcc
     3121 ttccctttct gtgtagcagt cttgaaatgt tctgtttaaa tcagtaggct taatgtgtta
     3181 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3241 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3301 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3361 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     3421 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3481 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3541 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     3601 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3661 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3721 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3781 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3841 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3901 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3961 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4021 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4081 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4141 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4201 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4261 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4321 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4381 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4441 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4501 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4561 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4621 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4681 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4741 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4801 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4861 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4921 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4981 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5041 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5101 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5161 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5221 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5281 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5341 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     5401 atcacgaggc cctttcgtc
//
