LOCUS       sequence-189663-        5676 bp    DNA     circular SYN 14-APR-2025
DEFINITION  MMEJ 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 5676)
  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 5676)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 MMEJ donor_U6_sgRNA_EF1a_GFP_polyA
FEATURES             Location/Qualifiers
     source          1..5676
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             218..274
                     /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             275..985
                     /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(420..438)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     688..707
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        1043..1291
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1043..1063
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1214..1233
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1313..1388
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1446..1657
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1670..1938
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      1966..1975
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1972..1974,1975..1977,1978..2691)
                     /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             join(1972..1974,1975..1977,1978..2688)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2017..2038)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2278..2297)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2625..2646
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2694..2918
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2939..3079
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2939..3068
                     /label=AAV2 ITR
     rep_origin      3154..3609
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3241..3260)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3451..3472
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3626..3645)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3745..3767
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3805..3823)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3891..3995
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3996..4064,4065..4856)
                     /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     complement(4214..4233)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5027..5615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5516..5535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccatcg atgccaagct tggtaccagt ccgcctagaa gcacttgcgg
      181 cccctccatc gtgcaccgca agtgcttctt aattaacgcc actaacttct ccctgttgaa
      241 acaagcaggg gatgtcgaag agaatcccgg gccaatggtg agcaagggcg aggaggataa
      301 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
      361 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
      421 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
      481 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
      541 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
      601 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
      661 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
      721 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
      781 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
      841 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
      901 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
      961 cggcatggac gagctgtaca agtaaggcgc gccgcggact gttactgagc tgcccgcaag
     1021 tgcttctagg cggactctcg aggagggcct atttcccatg attccttcat atttgcatat
     1081 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
     1141 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     1201 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     1261 tttatatatc ttgtggaaag gacgaaacac cgagtccgcc tagaagcact tggttttaga
     1321 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     1381 gtcggtgctt ttttgtttta gagctaggct agcaaggatc tgcgatcgct ccggtgcccg
     1441 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     1501 ttgaacgggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     1561 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     1621 cgttcttttt cgcaacgggt ttgccgccag aacacagctg aagcttcgag gggctcgcat
     1681 ctctccttca cgcgcccgcc gccctacctg aggccgccat ccacgccggt tgagtcgcgt
     1741 tctgccgcct cccgcctgtg gtgcctcctg aactgcgtcc gccgtctagg taagtttaaa
     1801 gctcaggtcg agaccgggcc tttgtccggc gctcccttgg agcctaccta gactcagccg
     1861 gctctccacg ctttgcctga ccctgcttgc tcaactctac gtctttgttt cgttttctgt
     1921 tctgcgccgt tacagatcca agctgtgacc ggcgcctaca ccggtgccac catggtgagc
     1981 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2041 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2101 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2161 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2221 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2281 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2341 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2401 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2461 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2521 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2581 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2641 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta agactgtgcc
     2701 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2761 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2821 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2881 caatagcagg catgctgggg atgcggtggg ctctatggct tgcggccgcg tcggccgcag
     2941 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     3001 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     3061 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3121 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     3181 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     3241 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     3301 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     3361 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3421 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3481 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     3541 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     3601 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3661 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3721 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3781 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3841 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3901 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3961 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     4021 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     4081 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     4141 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     4201 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     4261 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     4321 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     4381 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4441 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4501 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4561 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4621 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     4681 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4741 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4801 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4861 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4921 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4981 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5041 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5101 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5161 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5221 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5281 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5341 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5401 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5461 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5521 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5581 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5641 ggttcctggc cttttgctgg ccttttgctc acatgt
//
