LOCUS       sequence-452035-        7324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1 targeting donor for the insertion of Puro and a strong EF1a 
            promoter expressing mScarlet. To be co-transfected with sgRNA 
            plasmid px330-AAVS1 (Addgene #227272).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7324)
  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 7324)
  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-AAVS1-Ef1a-Puro-mScarlet
FEATURES             Location/Qualifiers
     source          1..7324
                     /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"
     misc_feature    402..1205
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    1212..1237
                     /label=SA
                     /note="splice acceptor site"
     CDS             1267..1323
                     /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             1339..1938
                     /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(1339..1358)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1835..1855
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1960..2081
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1997..2016)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2051..2070
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2100..2311
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          2312..3250
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3207..3227
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3288..3983
                     /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(3421..3439)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3689..3708
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4009..4233
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    4248..5084
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(5103..5119)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5103..5119)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5116..5138)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5127..5143
                     /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(5151..5157,5158..5175,5176..5181))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5196..5217
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5334..5351)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5505..6093)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5585..5604)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6264..7055,7056..7124))
                     /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     6887..6906
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7125..7229)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7297..7315
                     /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 ctgctttctc tgaccagcat
      421 tctctcccct gggcctgtgc cgctttctgt ctgcagcttg tggcctgggt cacctctacg
      481 gctggcccag atccttccct gccgcctcct tcaggttccg tcttcctcca ctccctcttc
      541 cccttgctct ctgctgtgtt gctgcccaag gatgctcttt ccggagcact tccttctcgg
      601 cgctgcacca cgtgatgtcc tctgagcgga tcctccccgt gtctgggtcc tctccgggca
      661 tctctcctcc ctcacccaac cccatgccgt cttcactcgc tgggttccct tttccttctc
      721 cttctggggc ctgtgccatc tctcgtttct taggatggcc ttctccgacg gatgtctccc
      781 ttgcgtcccg cctccccttc ttgtaggcct gcatcatcac cgtttttctg gacaacccca
      841 aagtaccccg tctccctggc tttagccacc tctccatcct cttgctttct ttgcctggac
      901 accccgttct cctgtggatt cgggtcacct ctcactcctt tcatttgggc agctccccta
      961 ccccccttac ctctctagtc tgtgctagct cttccagccc cctgtcatgg catcttccag
     1021 gggtccgaga gctcagctag tcttcttcct ccaacccggg cccctatgtc cacttcagga
     1081 cagcatgttt gctgcctcca gggatcctgt gtccccgagc tgggaccacc ttatattccc
     1141 agggccggtt aatgtggctc tggttctggg tacttttatc tgtcccctcc accccacagt
     1201 ggggctgtac actgacctct tctcttcctc ccacagggca tgcattctgg tggcggagga
     1261 tccggagcta ctaacttcag cctgctgaag caggccggag acgtggagga gaaccctgga
     1321 ccttctagag gaagcggaat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     1381 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     1441 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     1501 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     1561 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     1621 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     1681 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     1741 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     1801 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     1861 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     1921 cgcaagcccg gtgcctgagg cgcgcctatt atggtaccta acttgtttat tgcagcttat
     1981 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     2041 cattctagtt gtggtttgtc caaactcatc aatgtatctt acatatgcca tggatcgcgg
     2101 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
     2161 cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
     2221 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
     2281 ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc
     2341 tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct ggctgcagta
     2401 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc
     2461 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg
     2521 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat
     2581 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc
     2641 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg
     2701 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg
     2761 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat
     2821 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg
     2881 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg
     2941 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg
     3001 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag
     3061 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg
     3121 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct
     3181 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct
     3241 tccatttcag gtgtcgtgaa agggcgcatt cgacatcgat actagtaatg gtgagcaagg
     3301 gcgaggcagt gatcaaggag ttcatgcggt tcaaggtgca catggagggc tccatgaacg
     3361 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     3421 ccaagctgaa ggtgaccaag ggtggccccc tgcccttctc ctgggacatc ctgtcccctc
     3481 agttcatgta cggctccagg gccttcacca agcaccccgc cgacatcccc gactactata
     3541 agcagtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
     3601 ccgtgaccgt gacccaggac acctccctgg aggacggcac cctgatctac aaggtgaagc
     3661 tccgcggcac caacttccct cctgacggcc ccgtaatgca gaagaagaca atgggctggg
     3721 aagcgtccac cgagcggttg taccccgagg acggcgtgct gaagggcgac attaagatgg
     3781 ccctgcgcct gaaggacggc ggccgctacc tggcggactt caagaccacc tacaaggcca
     3841 agaagcccgt gcagatgccc ggcgcctaca acgtcgaccg caagttggac atcacctccc
     3901 acaacgagga ctacaccgtg gtggaacagt acgaacgctc cgagggccgc cactccaccg
     3961 gcggcatgga cgagctgtac aagtgatcgg tcgcctgcag gacgaccgct gtgccttcta
     4021 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     4081 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     4141 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     4201 gcaggcatgc tggggatgcg gtgggctcta tgggcggccg cgtcgacact agggacagga
     4261 ttggtgacag aaaagcccca tccttaggcc tcctccttcc tagtctcctg atattgggtc
     4321 taacccccac ctcctgttag gcagattcct tatctggtga cacaccccca tttcctggag
     4381 ccatctctct ccttgccaga acctctaagg tttgcttacg atggagccag agaggatcct
     4441 gggagggaga gcttggcagg gggtgggagg gaaggggggg atgcgtgacc tgcccggttc
     4501 tcagtggcca ccctgcgcta ccctctccca gaacctgagc tgctctgacg cggctgtctg
     4561 gtgcgtttca ctgatcctgg tgctgcagct tccttacact tcccaagagg agaagcagtt
     4621 tggaaaaaca aaatcagaat aagttggtcc tgagttctaa ctttggctct tcacctttct
     4681 agtccccaat ttatattgtt cctccgtgcg tcagttttac ctgtgagata aggccagtag
     4741 ccagccccgt cctggcaggg ctgtggtgag gaggggggtg tccgtgtgga aaactccctt
     4801 tgtgagaatg gtgcgtccta ggtgttcacc aggtcgtggc cgcctctact ccctttctct
     4861 ttctccatcc ttctttcctt aaagagtccc cagtgctatc tgggacatat tcctccgccc
     4921 agagcagggt cccgcttccc taaggccctg ctctgggctt ctgggtttga gtccttggca
     4981 agcccaggag aggcgctcag gcttccctgt cccccttcct cgtccaccat ctcatgcccc
     5041 tggctctcct gccccttccc tacaggggtt cctggctctg ctctaagctt ggcgtaatca
     5101 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5161 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5221 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5281 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5341 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5401 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5461 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5521 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5581 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5641 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5701 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5761 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5821 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5881 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5941 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6001 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6061 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6121 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6181 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6241 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6301 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6361 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6421 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6481 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6541 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6601 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6661 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6721 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6781 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6841 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6901 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6961 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7021 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7081 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7141 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7201 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     7261 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     7321 cgtc
//
