LOCUS       sequence-415028-        5989 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Puro-2A-mScarlet insertion into the N-terminus of
            the ACTB locus for actin visualization. To be co-transfected with 
            sgRNA plasmid px330-PITCh-ACTB Addgene #207748.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5989)
  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 5989)
  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-Puro-mScarlet-ACTB
FEATURES             Location/Qualifiers
     source          1..5989
                     /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(1401..1434)
                     /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             1443..2039
                     /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(1443..1462)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1939..1959
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2049..2105
                     /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(2112..2145)
                     /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             2154..2180
                     /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             2154..2180
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2184..2210
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2220..2915
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2353..2371)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2621..2640
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3768..3784)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3768..3784)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3781..3803)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3792..3808
                     /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(3816..3822,3823..3840,3841..3846))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3861..3882
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3999..4016)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4170..4758)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4250..4269)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4929..5720,5721..5789))
                     /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     5552..5571
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5790..5894)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5962..5980
                     /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 ccctataaaa cccagcggcg
      421 cgacgcgcca ccaccgccga gaccgcgtcc gccccgcgag cacagagcct cgcctttgcc
      481 gatccgccgc ccgtccacac ccgccgccag gtaagcccgg ccagccgacc ggggcaggcg
      541 gctcacggcc cggccgcagg cggccgcggc cccttcgccc gtgcagagcc gccgtctggg
      601 ccgcagcggg gggcgcatgg ggggggaacc ggaccgccgt ggggggcgcg ggagaagccc
      661 ctgggcctcc ggagatgggg gacaccccac gccagttcgg aggcgcgagg ccgcgctcgg
      721 gaggcgcgct ccgggggtgc cgctctcggg gcgggggcaa ccggcggggt ctttgtctga
      781 gccgggctct tgccaatggg gatcgcaggg tgggcgcggc ggagcccccg ccaggcccgg
      841 tgggggctgg ggcgccattg cgcgtgcgcg ctggtccttt gggcgctaac tgcgtgcgcg
      901 ctgggaattg gcgctaattg cgcgtgcgcg ctgggactca aggcgctaac tgcgcgtgcg
      961 ttctggggcc cggggtgccg cggcctgggc tggggcgaag gcgggctcgg ccggaagggg
     1021 tggggtcgcc gcggctcccg ggcgcttgcg cgcacttcct gcccgagccg ctggccgccc
     1081 gagggtgtgg ccgctgcgtg cgcgcgcgcc gacccggcgc tgtttgaacc gggcggaggc
     1141 ggggctggcg cccggttggg agggggttgg ggcctggctt cctgccgcgc gccgcgggga
     1201 cgcctccgac cagtgtttgc cttttatggt aataacgcgg ccggcccggc ttcctttgtc
     1261 cccaatctgg gcgcgcgccg gcgccccctg gcggcctaag gactcggcgc gccggaagtg
     1321 gccagggcgg gggcgacctc ggctcacagc gcgcccggct attctcgcaa ctgacaatgg
     1381 gcaagagcaa gaccggtagc taccgttcgt atagcataca ttatacgaag ttatctgcta
     1441 gcatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     1501 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     1561 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     1621 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     1681 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     1741 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     1801 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     1861 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     1921 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     1981 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg
     2041 gatccggagc tactaacttc agcctgctga agcaggccgg agacgtggag gagaaccctg
     2101 gaccttctag aataacttcg tatagcatac attatacgaa cggtagcacg cgttacccct
     2161 acgacgtgcc cgactacgcc ggctatccgt atgatgtccc ggactatgca ggaagcggaa
     2221 tggtgagcaa gggcgaggca gtgatcaagg agttcatgcg gttcaaggtg cacatggagg
     2281 gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     2341 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc tcctgggaca
     2401 tcctgtcccc tcagttcatg tacggctcca gggccttcac caagcacccc gccgacatcc
     2461 ccgactacta taagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     2521 aggacggcgg cgccgtgacc gtgacccagg acacctccct ggaggacggc accctgatct
     2581 acaaggtgaa gctccgcggc accaacttcc ctcctgacgg ccccgtaatg cagaagaaga
     2641 caatgggctg ggaagcgtcc accgagcggt tgtaccccga ggacggcgtg ctgaagggcg
     2701 acattaagat ggccctgcgc ctgaaggacg gcggccgcta cctggcggac ttcaagacca
     2761 cctacaaggc caagaagccc gtgcagatgc ccggcgccta caacgtcgac cgcaagttgg
     2821 acatcacctc ccacaacgag gactacaccg tggtggaaca gtacgaacgc tccgagggcc
     2881 gccactccac cggcggcatg gacgagctgt acaagctcga gggaggaagc ggaggatctg
     2941 gcggtaccga tgatgatatc gcagcgctcg tcgtcgacaa cggctccggc atgtgcaagg
     3001 ccggcttcgc gggcgacgat gccccccggg ccgtcttccc ctccatcgtg gggcgcccca
     3061 ggcaccaggt aggggagctg gctgggtggg gcagccccgg gagcgggcgg gaggcaaggg
     3121 cgctttctct gcacaggagc ctcccggttt ccggggtggg ggctgcgccc gtgctcaggg
     3181 cttcttgtcc tttccttccc agggcgtgat ggtgggcatg ggtcagaagg attcctatgt
     3241 gggcgacgag gcccagagca agagaggcat cctcaccctg aagtacccca tcgagcacgg
     3301 catcgtcacc aactgggacg acatggagaa aatctggcac cacaccttct acaatgagct
     3361 gcgtgtggct cccgaggagc accccgtgct gctgaccgag gcccccctga accccaaggc
     3421 caaccgcgag aagatgaccc aggtgagtgg cccgctacct cttctggtgg ccgcctccct
     3481 ccttcctggc ctcccggagc tgcgcccttt ctcactggtt ctctcttctg ccgttttccg
     3541 taggactctc ttctctgacc tgagtctcct ttggaactct gcaggttcta tttgcttttt
     3601 cccagatgag ctctttttct ggtgtttgtc tctctgacta ggtgtctaag acagtgttgt
     3661 gggtgtaggt actaacactg gctcgtgtga caaggccatg aggctggtgt aaagcggcct
     3721 tggagtgtgt attaagtagg cgcacagtaa agcttggcgt aatcatggtc atagctgttt
     3781 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3841 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3901 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3961 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4021 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4081 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4141 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4201 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4261 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4321 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4381 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4441 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4501 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4561 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4621 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4681 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4741 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4801 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4861 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4921 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4981 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5041 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5101 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5161 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5221 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5281 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5341 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5401 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5461 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5521 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5581 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5641 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5701 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5761 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5821 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5881 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     5941 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
