LOCUS       sequence-452027-        5753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty AAVS1 targeting donor for the insertion of Zeo 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 5753)
  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 5753)
  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-Zeo
FEATURES             Location/Qualifiers
     source          1..5753
                     /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..1713
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1735..1856
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1772..1791)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1826..1845
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1875..2374
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1910..1933)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2307..2326
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     polyA_signal    2438..2662
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    2677..3513
                     /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(3532..3548)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3532..3548)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3545..3567)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3556..3572
                     /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(3580..3586,3587..3604,3605..3610))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3625..3646
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3763..3780)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3934..4522)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4014..4033)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4693..5484,5485..5553))
                     /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     5316..5335
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5554..5658)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5726..5744
                     /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 ggccaagttg accagtgccg ttccggtgct caccgcgcgc
     1381 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttcagccg ggacttcgtg
     1441 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
     1501 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
     1561 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
     1621 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
     1681 tgcgtgcact tcgtggccga ggagcaggac tgaggcgcgc ctattatggt acctaacttg
     1741 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     1801 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttacata
     1861 tgccatggat cgcggggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt
     1921 agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac
     1981 atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac
     2041 tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca
     2101 aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa
     2161 gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca
     2221 gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg
     2281 cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc
     2341 gctgttctcc tcttcctcat ctccgggcct ttcggcgtcg tgaaagggcg cattcgacat
     2401 cgatactagt acgcgtcggt cgcctgcagg acgaccgctg tgccttctag ttgccagcca
     2461 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2521 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2581 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2641 ggggatgcgg tgggctctat gggcggccgc gtcgacacta gggacaggat tggtgacaga
     2701 aaagccccat ccttaggcct cctccttcct agtctcctga tattgggtct aacccccacc
     2761 tcctgttagg cagattcctt atctggtgac acacccccat ttcctggagc catctctctc
     2821 cttgccagaa cctctaaggt ttgcttacga tggagccaga gaggatcctg ggagggagag
     2881 cttggcaggg ggtgggaggg aaggggggga tgcgtgacct gcccggttct cagtggccac
     2941 cctgcgctac cctctcccag aacctgagct gctctgacgc ggctgtctgg tgcgtttcac
     3001 tgatcctggt gctgcagctt ccttacactt cccaagagga gaagcagttt ggaaaaacaa
     3061 aatcagaata agttggtcct gagttctaac tttggctctt cacctttcta gtccccaatt
     3121 tatattgttc ctccgtgcgt cagttttacc tgtgagataa ggccagtagc cagccccgtc
     3181 ctggcagggc tgtggtgagg aggggggtgt ccgtgtggaa aactcccttt gtgagaatgg
     3241 tgcgtcctag gtgttcacca ggtcgtggcc gcctctactc cctttctctt tctccatcct
     3301 tctttcctta aagagtcccc agtgctatct gggacatatt cctccgccca gagcagggtc
     3361 ccgcttccct aaggccctgc tctgggcttc tgggtttgag tccttggcaa gcccaggaga
     3421 ggcgctcagg cttccctgtc ccccttcctc gtccaccatc tcatgcccct ggctctcctg
     3481 ccccttccct acaggggttc ctggctctgc tctaagcttg gcgtaatcat ggtcatagct
     3541 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3601 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3661 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3721 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3781 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3841 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3901 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3961 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4021 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4081 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4141 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4201 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4261 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4321 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4381 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4441 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4501 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4561 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4621 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4681 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4741 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4801 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4861 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4921 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4981 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5041 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5101 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5161 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5221 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5281 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5341 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5401 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5461 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5521 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5581 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5641 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     5701 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtc
//
