LOCUS       sequence-452031-        7345 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAVS1 targeting donor for the insertion of Puro and a strong EF1a 
            promoter 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 7345)
  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 7345)
  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-moxGFP
FEATURES             Location/Qualifiers
     source          1..7345
                     /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             join(3288..3290,3291..3293,3294..4007)
                     /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(3333..3354)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3941..3962
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4030..4254
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    4269..5105
                     /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(5124..5140)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5124..5140)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5137..5159)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5148..5164
                     /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(5172..5178,5179..5196,5197..5202))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5217..5238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5355..5372)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5526..6114)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5606..5625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6285..7076,7077..7145))
                     /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     6908..6927
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7146..7250)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7318..7336
                     /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 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtgaacg
     3361 gccacaagtt ctccgtgcgg ggcgagggcg agggcgatgc caccaacggc aagctgaccc
     3421 tgaagttcat cagcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     3481 tgacctacgg cgtgcagagc ttctcccgct accccgacca catgaagcgc cacgacttct
     3541 tcaagagcgc catgcccgaa ggctacgtcc aggagcgcac catctccttc aaggacgacg
     3601 gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     3661 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     3721 acttcaactc ccacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca
     3781 acttcaagat ccgccataac gtggaggacg gctccgtgca gctcgccgac cactaccagc
     3841 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgtccaccc
     3901 agtccaagct gtccaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     3961 tgaccgccgc cgggatcact cacggcatgg acgagctgta caagtgatcg gtcgcctgca
     4021 ggacgaccgc tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     4081 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     4141 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     4201 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atgggcggcc
     4261 gcgtcgacac tagggacagg attggtgaca gaaaagcccc atccttaggc ctcctccttc
     4321 ctagtctcct gatattgggt ctaaccccca cctcctgtta ggcagattcc ttatctggtg
     4381 acacaccccc atttcctgga gccatctctc tccttgccag aacctctaag gtttgcttac
     4441 gatggagcca gagaggatcc tgggagggag agcttggcag ggggtgggag ggaagggggg
     4501 gatgcgtgac ctgcccggtt ctcagtggcc accctgcgct accctctccc agaacctgag
     4561 ctgctctgac gcggctgtct ggtgcgtttc actgatcctg gtgctgcagc ttccttacac
     4621 ttcccaagag gagaagcagt ttggaaaaac aaaatcagaa taagttggtc ctgagttcta
     4681 actttggctc ttcacctttc tagtccccaa tttatattgt tcctccgtgc gtcagtttta
     4741 cctgtgagat aaggccagta gccagccccg tcctggcagg gctgtggtga ggaggggggt
     4801 gtccgtgtgg aaaactccct ttgtgagaat ggtgcgtcct aggtgttcac caggtcgtgg
     4861 ccgcctctac tccctttctc tttctccatc cttctttcct taaagagtcc ccagtgctat
     4921 ctgggacata ttcctccgcc cagagcaggg tcccgcttcc ctaaggccct gctctgggct
     4981 tctgggtttg agtccttggc aagcccagga gaggcgctca ggcttccctg tcccccttcc
     5041 tcgtccacca tctcatgccc ctggctctcc tgccccttcc ctacaggggt tcctggctct
     5101 gctctaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5161 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5221 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5281 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5341 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5401 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5461 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5521 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5581 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5641 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5701 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5761 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5821 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5881 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5941 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6001 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6061 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6121 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6181 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6241 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6301 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6361 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6421 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6481 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6541 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6601 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6661 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6721 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6781 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6841 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6901 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6961 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7021 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7081 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7141 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7201 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7261 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     7321 aggcgtatca cgaggccctt tcgtc
//
