LOCUS       sequence-452017-        6428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty AAVS1 targeting donor for the insertion of Blast and a strong 
            EF1a 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 6428)
  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 6428)
  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-Blast
FEATURES             Location/Qualifiers
     source          1..6428
                     /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..2110
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          2111..3049
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3006..3026
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     polyA_signal    3113..3337
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3352..4188
                     /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(4207..4223)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4207..4223)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4220..4242)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4231..4247
                     /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(4255..4261,4262..4279,4280..4285))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4300..4321
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4438..4455)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4609..5197)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4689..4708)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5368..6159,6160..6228))
                     /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     5991..6010
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6229..6333)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6401..6419
                     /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 gcagagcgca catcgcccac
     1921 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
     1981 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
     2041 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
     2101 cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg
     2161 cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct
     2221 tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg
     2281 tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct
     2341 tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc
     2401 tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg
     2461 tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc
     2521 ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg
     2581 ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag
     2641 gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc
     2701 agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca
     2761 aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc
     2821 gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg
     2881 ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc
     2941 agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt
     3001 cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaaa
     3061 gggcgcattc gacatcgata ctagtacgcg tcggtcgcct gcaggacgac cgctgtgcct
     3121 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3181 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3241 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3301 aatagcaggc atgctgggga tgcggtgggc tctatgggcg gccgcgtcga cactagggac
     3361 aggattggtg acagaaaagc cccatcctta ggcctcctcc ttcctagtct cctgatattg
     3421 ggtctaaccc ccacctcctg ttaggcagat tccttatctg gtgacacacc cccatttcct
     3481 ggagccatct ctctccttgc cagaacctct aaggtttgct tacgatggag ccagagagga
     3541 tcctgggagg gagagcttgg cagggggtgg gagggaaggg ggggatgcgt gacctgcccg
     3601 gttctcagtg gccaccctgc gctaccctct cccagaacct gagctgctct gacgcggctg
     3661 tctggtgcgt ttcactgatc ctggtgctgc agcttcctta cacttcccaa gaggagaagc
     3721 agtttggaaa aacaaaatca gaataagttg gtcctgagtt ctaactttgg ctcttcacct
     3781 ttctagtccc caatttatat tgttcctccg tgcgtcagtt ttacctgtga gataaggcca
     3841 gtagccagcc ccgtcctggc agggctgtgg tgaggagggg ggtgtccgtg tggaaaactc
     3901 cctttgtgag aatggtgcgt cctaggtgtt caccaggtcg tggccgcctc tactcccttt
     3961 ctctttctcc atccttcttt ccttaaagag tccccagtgc tatctgggac atattcctcc
     4021 gcccagagca gggtcccgct tccctaaggc cctgctctgg gcttctgggt ttgagtcctt
     4081 ggcaagccca ggagaggcgc tcaggcttcc ctgtccccct tcctcgtcca ccatctcatg
     4141 cccctggctc tcctgcccct tccctacagg ggttcctggc tctgctctaa gcttggcgta
     4201 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4261 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4321 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4381 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4441 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4501 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4561 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4621 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4681 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4741 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4801 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4861 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4921 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4981 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5041 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5101 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5161 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5221 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5281 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5341 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5401 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5461 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5521 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5581 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5641 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5701 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5761 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5821 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5881 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5941 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6001 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6061 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6121 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6181 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6241 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6301 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6361 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     6421 ctttcgtc
//
