LOCUS       sequence-189989-        7181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HR donor for fusing a p2A-mCherry reporter to mouse Actb. EGFP 
            driven by EF1a promoter and U6-driven sgRNAs targeting Actb. AAV 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7181)
  AUTHORS   Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, Shen X, Wei Y, Huang Z, 
            Ying W, Wang Y, Nie YH, Zhang CC, Li S, Cheng L, Wang Q, Wu Y, Huang
            P, Sun Q, Shi L, Yang H
  TITLE     Homology-mediated end joining-based targeted integration using 
            CRISPR/Cas9.
  JOURNAL   Cell Res. 2017 May 19. doi: 10.1038/cr.2017.76.
  PUBMED    28524166
REFERENCE   2  (bases 1 to 7181)
  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: "Cell Res. 
            2017 May 19. doi: 10.1038/cr.2017.76."
COMMENT     Sequence Label: AAV_Actb HR donor_U6_sgRNA_EF1a_GFP_polyA
FEATURES             Location/Qualifiers
     source          1..7181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(274..984)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(552..571)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     821..839
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(985..1041)
                     /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"
     repeat_region   1878..2007
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2069..2657)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2149..2168)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2828..3619,3620..3688))
                     /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     3451..3470
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3689..3793)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3861..3879
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3917..3939)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4039..4058
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4075..4530)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4212..4233)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4424..4443
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   4605..4745
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4616..4745
                     /label=AAV2 ITR
     polyA_signal    4766..4990
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(join(4993..5706,5707..5709,5710..5712))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(4996..5706,5707..5709,5710..5712))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5038..5059)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5387..5406
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5646..5667
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      5709..5718
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     LTR             5746..6014
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     promoter        complement(6027..6238)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(6296..6371)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(6393..6641)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(6451..6470)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(6621..6641)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gtcctcagcc acatttgtag aactttgggg gatgtttgct ccaaccaact gctgtcgcct
       61 tcaccgttcc agtttttaaa tcctgagtca aaagcgccaa aacaaaacaa aaaaacttaa
      121 aaaaaaaaaa aaaaaaaaaa aaaaaacttt aaaaaaaatt taaaaaaaca aagccatgcc
      181 aatgttgtct cttatttttt ttttttctgc gcaagttagg ttttgtcaaa gaaagggtgt
      241 aaaacgcagc tcagtaacag tccgcggcgc gccttacttg tacagctcgt ccatgccgcc
      301 ggtggagtgg cggccctcgg cgcgttcgta ctgttccacg atggtgtagt cctcgttgtg
      361 ggaggtgatg tccaacttga tgttgacgtt gtaggcgccg ggcagctgca cgggcttctt
      421 ggccttgtag gtggtcttga cctcagcgtc gtagtggccg ccgtccttca gcttcagcct
      481 ctgcttgatc tcgcccttca gggcgccgtc ctcggggtac atccgctcgg aggaggcctc
      541 ccagcccatg gtcttcttct gcattacggg gccgtcggag gggaagttgg tgccgcgcag
      601 cttcaccttg tagatgaact cgccgtcctg cagggaggag tcctgggtca cggtcaccac
      661 gccgccgtcc tcgaagttca tcacgcgctc ccacttgaag ccctcgggga aggacagctt
      721 caagtagtcg gggatgtcgg cggggtgctt cacgtaggcc ttggagccgt acatgaactg
      781 aggggacagg atgtcccagg cgaagggcag ggggccaccc ttggtcacct tcagcttggc
      841 ggtctgggtg ccctcgtagg ggcggccctc gccctcgccc tcgatctcga actcgtggcc
      901 gttcacggag ccctccatgt gcaccttgaa gcgcatgaac tccttgatga tggccatgtt
      961 atcctcctcg cccttgctca ccattggccc gggattctct tcgacatccc ctgcttgttt
     1021 caacagggag aagttagtgg cgttaattaa gaagcacttg cggtgcacga tggaggggcc
     1081 ggactcatcg tactcctgct tgctgatcca catctgctgg aaggtggaca gtgaggccag
     1141 gatggagcca ccgatccaca cagagtactt gcgctcagga ggagcaatga tctgaagaaa
     1201 gcaagacaag atggtgaatg gtgagctctc tgggtgctgg gattcccctt aaacctagaa
     1261 ggttgctctg acaaccacag ggcccaccct caccaagcta aggatgctta gcttaccttg
     1321 atcttcatgg tgctaggagc cagagcagta atctccttct gcatcctgtc agcaatgcct
     1381 gggtacatgg tggtaccacc agacagcact gtgttggcat agaggtcttt acggatgtca
     1441 acgtcacact tcatgatgga attgaatgta gtttcatgga tgccacagga ttccatacct
     1501 aagagaagag tgacagaaat cagacttaac ttgtactatg gcctcaggag ttttgtcacc
     1561 tttagatgga gaaaggacta ggctacaact tacccaagaa ggaaggctgg aaaagagcct
     1621 cagggcatcg gaaccgctcg ttgccaatag tgatgacctg gccgtcaggc agctcatagc
     1681 tcttctccag ggaggaagag gatgcggcag tggccatctc ctgctcgaag tctagagcaa
     1741 catagcacag cttctctttg atgtcacgca cgatttccct ctcagctgtg gtggtgaagc
     1801 tgtagccacg ctcggtcagg atcttcatga ggtagtctgt caggtcccgg ggtaccaagc
     1861 ttggcatcga tggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     1921 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggcg gcctcagtga
     1981 gcgagcgagc gcgcagctgc ctgcaggaca tgtgagcaaa aggccagcaa aaggccagga
     2041 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2101 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2161 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2221 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2281 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2341 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2401 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2461 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2521 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2581 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2641 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2701 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2761 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2821 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2881 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2941 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3001 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3061 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3121 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3181 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3241 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3301 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3361 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3421 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3481 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3541 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3601 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3661 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3721 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3781 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     3841 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     3901 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3961 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     4021 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac cataaaattg
     4081 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     4141 accaatagac cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagagt
     4201 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     4261 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca cccaaatcaa
     4321 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     4381 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     4441 gagcgggcgc taaggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     4501 ccgcgcttaa tgcgccgcta cagggcgcgt actatggttg ctttgacgta tgcggtgtga
     4561 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgcccctgca ggcagctgcg
     4621 cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg
     4681 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg
     4741 ttcctgcggc cgacgcggcc gcaagccata gagcccaccg catccccagc atgcctgcta
     4801 ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga
     4861 cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg
     4921 gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta
     4981 gaaggcacag tcttacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac
     5041 gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg
     5101 ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg
     5161 ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt
     5221 caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt
     5281 gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc
     5341 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc
     5401 gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa
     5461 gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag
     5521 ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag
     5581 ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc
     5641 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc
     5701 cttgctcacc atggtggcac cggtgtaggc gccggtcaca gcttggatct gtaacggcgc
     5761 agaacagaaa acgaaacaaa gacgtagagt tgagcaagca gggtcaggca aagcgtggag
     5821 agccggctga gtctaggtag gctccaaggg agcgccggac aaaggcccgg tctcgacctg
     5881 agctttaaac ttacctagac ggcggacgca gttcaggagg caccacaggc gggaggcggc
     5941 agaacgcgac tcaaccggcg tggatggcgg cctcaggtag ggcggcgggc gcgtgaagga
     6001 gagatgcgag cccctcgaag cttcagctgt gttctggcgg caaacccgtt gcgaaaaaga
     6061 acgttcacgg cgactactgc acttatatac ggttctcccc caccctcggg aaaaaggcgg
     6121 agccagtaca cgacatcact ttcccagttt accccgcgcc accttctcta ggcacccgtt
     6181 caattgccga cccctccccc caacttctcg gggactgtgg gcgatgtgcg ctctgcccac
     6241 tgacgggcac cggagcgatc gcagatcctt gctagcctag ctctaaaaca aaaaagcacc
     6301 gactcggtgc cactttttca agttgataac ggactagcct tattttaact tgctatttct
     6361 agctctaaaa ccaagtgctt ctaggcggac tcggtgtttc gtcctttcca caagatatat
     6421 aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa
     6481 cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt
     6541 actaatatct ttgtgtttac agtcaaatta attccaatta tctctctaac agccttgtat
     6601 cgtatatgca aatatgaagg aatcatggga aataggccct cctcgagagc cctcccacta
     6661 gataccatca gcaaggccta gcccacccta gatgcccccc ccaatagagc tccacaccca
     6721 cccaacctgc tccccacacc cagcaaagcc acagagccac aagctgtttg tgtaaggtaa
     6781 ggtgtgcact tttattggtc tcaagtcagt gtacaggcca gccctggctg cctcaacacc
     6841 tcaaccccct cccagggaga ccaaagcctt catacatcaa gttgggggga caaaaaaaag
     6901 ggaggcctca gacctgggcc attcagaaat taaaaacaaa aatgaagtat taaggcggaa
     6961 gatttaaaaa aatttttgca gtacataatt tacacagaag caatgctgtc accttccccg
     7021 gggtggactc agggcatgga cgcgaccatc ctcctcttag gagtgggggt ggcttttggg
     7081 agggtgaggg acttcctgta accacttatt tcatggatac ttggaatgac tattaaaaaa
     7141 agacaaaacc aaaaaaaaaa aaaaacaaaa caatgtacaa a
//
