LOCUS       sequence-452039-        6694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1 targeting donor for the insertion of Puro and a medium 
            strength PGK expressing moxGFP. 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 6694)
  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 6694)
  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-moxGFP
FEATURES             Location/Qualifiers
     source          1..6694
                     /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"
     CDS             join(2637..2639,2640..2642,2643..3356)
                     /codon_start=1
                     /product="monomeric GFP variant optimized for use in 
                     oxidizing environments such as the secretory pathway 
                     (Costantini et al., 2015)"
                     /label=moxGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2682..2703)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3290..3311
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3379..3603
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3618..4454
                     /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(4473..4489)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4473..4489)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4486..4508)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4497..4513
                     /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(4521..4527,4528..4545,4546..4551))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4566..4587
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4704..4721)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4875..5463)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4955..4974)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5634..6425,6426..6494))
                     /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     6257..6276
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6495..6599)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6667..6685
                     /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 ctagtaatgg
     2641 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2701 acgtgaacgg ccacaagttc tccgtgcggg gcgagggcga gggcgatgcc accaacggca
     2761 agctgaccct gaagttcatc agcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2821 tgaccaccct gacctacggc gtgcagagct tctcccgcta ccccgaccac atgaagcgcc
     2881 acgacttctt caagagcgcc atgcccgaag gctacgtcca ggagcgcacc atctccttca
     2941 aggacgacgg cacctacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     3001 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     3061 tggagtacaa cttcaactcc cacaacgtct atatcaccgc cgacaagcag aagaacggca
     3121 tcaaggccaa cttcaagatc cgccataacg tggaggacgg ctccgtgcag ctcgccgacc
     3181 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     3241 tgtccaccca gtccaagctg tccaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     3301 tggagttcgt gaccgccgcc gggatcactc acggcatgga cgagctgtac aagtgatcgg
     3361 tcgcctgcag gacgaccgct gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3421 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3481 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3541 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3601 tgggcggccg cgtcgacact agggacagga ttggtgacag aaaagcccca tccttaggcc
     3661 tcctccttcc tagtctcctg atattgggtc taacccccac ctcctgttag gcagattcct
     3721 tatctggtga cacaccccca tttcctggag ccatctctct ccttgccaga acctctaagg
     3781 tttgcttacg atggagccag agaggatcct gggagggaga gcttggcagg gggtgggagg
     3841 gaaggggggg atgcgtgacc tgcccggttc tcagtggcca ccctgcgcta ccctctccca
     3901 gaacctgagc tgctctgacg cggctgtctg gtgcgtttca ctgatcctgg tgctgcagct
     3961 tccttacact tcccaagagg agaagcagtt tggaaaaaca aaatcagaat aagttggtcc
     4021 tgagttctaa ctttggctct tcacctttct agtccccaat ttatattgtt cctccgtgcg
     4081 tcagttttac ctgtgagata aggccagtag ccagccccgt cctggcaggg ctgtggtgag
     4141 gaggggggtg tccgtgtgga aaactccctt tgtgagaatg gtgcgtccta ggtgttcacc
     4201 aggtcgtggc cgcctctact ccctttctct ttctccatcc ttctttcctt aaagagtccc
     4261 cagtgctatc tgggacatat tcctccgccc agagcagggt cccgcttccc taaggccctg
     4321 ctctgggctt ctgggtttga gtccttggca agcccaggag aggcgctcag gcttccctgt
     4381 cccccttcct cgtccaccat ctcatgcccc tggctctcct gccccttccc tacaggggtt
     4441 cctggctctg ctctaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4501 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4561 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4621 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4681 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4741 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4801 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4861 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4921 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4981 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5041 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5101 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5161 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5221 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5281 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5341 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5401 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5461 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5521 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5581 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5641 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5701 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5761 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5821 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5881 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5941 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6001 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6061 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6121 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6181 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6241 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6301 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6361 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6421 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6481 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6541 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6601 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     6661 tataaaaata ggcgtatcac gaggcccttt cgtc
//
