LOCUS       sequence-452020-        5978 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty AAVS1 targeting donor for the insertion of Puro and a medium 
            strength PGK promoter. A CDS can be cloned adjacent to the promoter 
            via restriction or gibson cloning using the MluI cut site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5978)
  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 5978)
  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-PGK-Puro
FEATURES             Location/Qualifiers
     source          1..5978
                     /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..2599
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2135..2158)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2532..2551
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     polyA_signal    2663..2887
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    2902..3738
                     /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(3757..3773)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3757..3773)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3770..3792)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3781..3797
                     /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(3805..3811,3812..3829,3830..3835))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3850..3871
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3988..4005)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4159..4747)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4239..4258)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4918..5709,5710..5778))
                     /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     5541..5560
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5779..5883)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5951..5969
                     /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 ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg
     2161 cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg
     2221 ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag
     2281 gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg
     2341 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
     2401 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg
     2461 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
     2521 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
     2581 ctcatctccg ggcctttcgg cgtcgtgaaa gggcgcattc gacatcgata ctagtacgcg
     2641 tcggtcgcct gcaggacgac cgctgtgcct tctagttgcc agccatctgt tgtttgcccc
     2701 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     2761 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     2821 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     2881 tctatgggcg gccgcgtcga cactagggac aggattggtg acagaaaagc cccatcctta
     2941 ggcctcctcc ttcctagtct cctgatattg ggtctaaccc ccacctcctg ttaggcagat
     3001 tccttatctg gtgacacacc cccatttcct ggagccatct ctctccttgc cagaacctct
     3061 aaggtttgct tacgatggag ccagagagga tcctgggagg gagagcttgg cagggggtgg
     3121 gagggaaggg ggggatgcgt gacctgcccg gttctcagtg gccaccctgc gctaccctct
     3181 cccagaacct gagctgctct gacgcggctg tctggtgcgt ttcactgatc ctggtgctgc
     3241 agcttcctta cacttcccaa gaggagaagc agtttggaaa aacaaaatca gaataagttg
     3301 gtcctgagtt ctaactttgg ctcttcacct ttctagtccc caatttatat tgttcctccg
     3361 tgcgtcagtt ttacctgtga gataaggcca gtagccagcc ccgtcctggc agggctgtgg
     3421 tgaggagggg ggtgtccgtg tggaaaactc cctttgtgag aatggtgcgt cctaggtgtt
     3481 caccaggtcg tggccgcctc tactcccttt ctctttctcc atccttcttt ccttaaagag
     3541 tccccagtgc tatctgggac atattcctcc gcccagagca gggtcccgct tccctaaggc
     3601 cctgctctgg gcttctgggt ttgagtcctt ggcaagccca ggagaggcgc tcaggcttcc
     3661 ctgtccccct tcctcgtcca ccatctcatg cccctggctc tcctgcccct tccctacagg
     3721 ggttcctggc tctgctctaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3781 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3841 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3901 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3961 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4021 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4081 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4141 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4201 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4261 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4321 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4381 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4441 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4501 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4561 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4621 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4681 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4741 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4801 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4861 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4921 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4981 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5041 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5101 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5161 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5221 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5281 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5341 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5401 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5461 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5521 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5581 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5641 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5701 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5761 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5821 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5881 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     5941 aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
