LOCUS       sequence-452091-        6022 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for Puro-2A-mStayGold 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 6022)
  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 6022)
  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-mStayGold-ACTB
FEATURES             Location/Qualifiers
     source          1..6022
                     /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             2169..2210
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2226..2903
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ando et al., 2023)"
                     /label=mStayGold(J)
                     /note="includes multiple mutations plus an N-terminal 
                     iinker sequence"
                     /translation="MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     primer_bind     complement(3801..3817)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3801..3817)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3814..3836)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3825..3841
                     /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(3849..3855,3856..3873,3874..3879))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3894..3915
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4032..4049)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4203..4791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4283..4302)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4962..5753,5754..5822))
                     /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     5585..5604
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5823..5927)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5995..6013
                     /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 cgtggaggtg
     2161 gcggttccgg caagcccatc cccaaccccc tgctgggcct ggacagcacc ggaggtggcg
     2221 gaagcatggt gtctacaggc gaggaactgt ttaccggcgt ggtgcccttc aagttccagc
     2281 tgaagggcac catcaacggc aagagcttca ccgtggaagg cgagggcgag ggcaatagcc
     2341 acgagggcag ccacaaaggc aagtatgtgt gcaccagcgg caaactgcca atgtcttggg
     2401 ccgccctggg aactagcttc ggctatggca tgaaatacta taccaagtac cccagcggcc
     2461 tgaaaaactg gttccacgag gtgatgcctg agggcttcac ctacgacaga cacatccagt
     2521 acaagggcga cggcagcatc cacgccaagc accagcactt catgaagaac ggcacctacc
     2581 acaacatcgt ggagttcacc ggccaggact tcaaggagaa cagccccgtg ctgaccggcg
     2641 acatggacgt gagcctgccc aacgaggtgc agcacatccc cattgatgac ggcgtggagt
     2701 gcacagtgac cctgcagtac cctctgctga gcgacgaaag caagtgcgtg gaagcctatc
     2761 agaacaccat catcaagccc ctgcacaatc agccagcccc cgatgtgcca tttcactgga
     2821 tcagaaagca gtacacccag agcaaggacg acaccgagga gagagaccac atcatccaga
     2881 gcgagaccct ggaggcccac ctgccttggc atgagcctag cgcaagcgca gtgggaggtg
     2941 gcggttccct cgagggagga agcggaggat ctggcggtac cgatgatgat atcgcagcgc
     3001 tcgtcgtcga caacggctcc ggcatgtgca aggccggctt cgcgggcgac gatgcccccc
     3061 gggccgtctt cccctccatc gtggggcgcc ccaggcacca ggtaggggag ctggctgggt
     3121 ggggcagccc cgggagcggg cgggaggcaa gggcgctttc tctgcacagg agcctcccgg
     3181 tttccggggt gggggctgcg cccgtgctca gggcttcttg tcctttcctt cccagggcgt
     3241 gatggtgggc atgggtcaga aggattccta tgtgggcgac gaggcccaga gcaagagagg
     3301 catcctcacc ctgaagtacc ccatcgagca cggcatcgtc accaactggg acgacatgga
     3361 gaaaatctgg caccacacct tctacaatga gctgcgtgtg gctcccgagg agcaccccgt
     3421 gctgctgacc gaggcccccc tgaaccccaa ggccaaccgc gagaagatga cccaggtgag
     3481 tggcccgcta cctcttctgg tggccgcctc cctccttcct ggcctcccgg agctgcgccc
     3541 tttctcactg gttctctctt ctgccgtttt ccgtaggact ctcttctctg acctgagtct
     3601 cctttggaac tctgcaggtt ctatttgctt tttcccagat gagctctttt tctggtgttt
     3661 gtctctctga ctaggtgtct aagacagtgt tgtgggtgta ggtactaaca ctggctcgtg
     3721 tgacaaggcc atgaggctgg tgtaaagcgg ccttggagtg tgtattaagt aggcgcacag
     3781 taaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3841 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3901 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3961 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4021 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4081 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4141 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4201 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4261 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4321 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4381 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4441 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4501 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4561 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4621 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4681 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4741 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4801 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4861 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4921 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4981 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5041 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5101 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5161 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5221 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5281 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5341 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5401 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5461 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5521 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5581 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5641 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5701 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5761 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5821 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     5881 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     5941 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     6001 cgtatcacga ggccctttcg tc
//
