LOCUS       sequence-451274-        6404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty AAVS1 targeting donor for the insertion of Zeo 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 6404)
  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 6404)
  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-Zeo
FEATURES             Location/Qualifiers
     source          1..6404
                     /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..2086
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          2087..3025
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2982..3002
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     polyA_signal    3089..3313
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3328..4164
                     /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(4183..4199)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4183..4199)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4196..4218)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4207..4223
                     /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(4231..4237,4238..4255,4256..4261))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4276..4297
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4414..4431)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4585..5173)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4665..4684)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5344..6135,6136..6204))
                     /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     5967..5986
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6205..6309)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6377..6395
                     /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 cgcggggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     1921 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     1981 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     2041 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
     2101 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
     2161 cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag
     2221 ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg cctggcctgg
     2281 gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga
     2341 taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga
     2401 tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg gggccgcggg
     2461 cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg
     2521 gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct
     2581 cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc
     2641 gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg
     2701 gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc
     2761 agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt
     2821 ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt
     2881 tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc
     2941 cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt
     3001 tcaaagtttt tttcttccat ttcaggtgtc gtgaaagggc gcattcgaca tcgatactag
     3061 tacgcgtcgg tcgcctgcag gacgaccgct gtgccttcta gttgccagcc atctgttgtt
     3121 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3181 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3241 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     3301 gtgggctcta tgggcggccg cgtcgacact agggacagga ttggtgacag aaaagcccca
     3361 tccttaggcc tcctccttcc tagtctcctg atattgggtc taacccccac ctcctgttag
     3421 gcagattcct tatctggtga cacaccccca tttcctggag ccatctctct ccttgccaga
     3481 acctctaagg tttgcttacg atggagccag agaggatcct gggagggaga gcttggcagg
     3541 gggtgggagg gaaggggggg atgcgtgacc tgcccggttc tcagtggcca ccctgcgcta
     3601 ccctctccca gaacctgagc tgctctgacg cggctgtctg gtgcgtttca ctgatcctgg
     3661 tgctgcagct tccttacact tcccaagagg agaagcagtt tggaaaaaca aaatcagaat
     3721 aagttggtcc tgagttctaa ctttggctct tcacctttct agtccccaat ttatattgtt
     3781 cctccgtgcg tcagttttac ctgtgagata aggccagtag ccagccccgt cctggcaggg
     3841 ctgtggtgag gaggggggtg tccgtgtgga aaactccctt tgtgagaatg gtgcgtccta
     3901 ggtgttcacc aggtcgtggc cgcctctact ccctttctct ttctccatcc ttctttcctt
     3961 aaagagtccc cagtgctatc tgggacatat tcctccgccc agagcagggt cccgcttccc
     4021 taaggccctg ctctgggctt ctgggtttga gtccttggca agcccaggag aggcgctcag
     4081 gcttccctgt cccccttcct cgtccaccat ctcatgcccc tggctctcct gccccttccc
     4141 tacaggggtt cctggctctg ctctaagctt ggcgtaatca tggtcatagc tgtttcctgt
     4201 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4261 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4321 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4381 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4441 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4501 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4561 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4621 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4681 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4741 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4801 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4861 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4921 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4981 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5041 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5101 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5161 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5221 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5281 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5341 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5401 catagttgcc tgacttcccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5461 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5521 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5581 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5641 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5701 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5761 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5821 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5881 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5941 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6001 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6061 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6121 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6181 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6241 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6301 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     6361 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc
//
