LOCUS       sequence-452024-        5777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty AAVS1 targeting donor for the insertion of Blast 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 5777)
  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 5777)
  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-Blast
FEATURES             Location/Qualifiers
     source          1..5777
                     /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..1737
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    1759..1880
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1796..1815)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1850..1869
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1899..2398
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1934..1957)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2331..2350
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     polyA_signal    2462..2686
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    2701..3537
                     /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(3556..3572)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3556..3572)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3569..3591)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3580..3596
                     /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(3604..3610,3611..3628,3629..3634))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3649..3670
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3787..3804)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3958..4546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4038..4057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4717..5508,5509..5577))
                     /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     5340..5359
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5578..5682)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5750..5768
                     /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 ggccaagcct ttgtctcaag aagaatccac cctcattgaa
     1381 agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca
     1441 gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga
     1501 ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact
     1561 tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga
     1621 caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag
     1681 ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctgaggc
     1741 gcgcctatta tggtacctaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     1801 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     1861 aaactcatca atgtatctta catatgccat ggatcgcggg gtaggggagg cgcttttccc
     1921 aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg
     1981 cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg
     2041 tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca
     2101 gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat
     2161 ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct
     2221 ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag
     2281 gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac
     2341 gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcggc
     2401 gtcgtgaaag ggcgcattcg acatcgatac tagtacgcgt cggtcgcctg caggacgacc
     2461 gctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2521 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2581 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2641 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgggcgg ccgcgtcgac
     2701 actagggaca ggattggtga cagaaaagcc ccatccttag gcctcctcct tcctagtctc
     2761 ctgatattgg gtctaacccc cacctcctgt taggcagatt ccttatctgg tgacacaccc
     2821 ccatttcctg gagccatctc tctccttgcc agaacctcta aggtttgctt acgatggagc
     2881 cagagaggat cctgggaggg agagcttggc agggggtggg agggaagggg gggatgcgtg
     2941 acctgcccgg ttctcagtgg ccaccctgcg ctaccctctc ccagaacctg agctgctctg
     3001 acgcggctgt ctggtgcgtt tcactgatcc tggtgctgca gcttccttac acttcccaag
     3061 aggagaagca gtttggaaaa acaaaatcag aataagttgg tcctgagttc taactttggc
     3121 tcttcacctt tctagtcccc aatttatatt gttcctccgt gcgtcagttt tacctgtgag
     3181 ataaggccag tagccagccc cgtcctggca gggctgtggt gaggaggggg gtgtccgtgt
     3241 ggaaaactcc ctttgtgaga atggtgcgtc ctaggtgttc accaggtcgt ggccgcctct
     3301 actccctttc tctttctcca tccttctttc cttaaagagt ccccagtgct atctgggaca
     3361 tattcctccg cccagagcag ggtcccgctt ccctaaggcc ctgctctggg cttctgggtt
     3421 tgagtccttg gcaagcccag gagaggcgct caggcttccc tgtccccctt cctcgtccac
     3481 catctcatgc ccctggctct cctgcccctt ccctacaggg gttcctggct ctgctctaag
     3541 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3601 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3661 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3721 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3781 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3841 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3901 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3961 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4021 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4081 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4141 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4201 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4261 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4321 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4381 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4441 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4501 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4561 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4621 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4681 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4741 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4801 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4861 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4921 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4981 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5041 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5101 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5161 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5221 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5281 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5341 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5401 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5461 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5521 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5581 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5641 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5701 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     5761 cacgaggccc tttcgtc
//
