LOCUS       sequence-192984-        5612 bp    DNA     circular SYN 15-APR-2025
DEFINITION  NHEJ 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 5612)
  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 5612)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 NHEJ donor_U6_sgRNA_EF1a_GFP_polyA
FEATURES             Location/Qualifiers
     source          1..5612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   27..156
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(218..806)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(298..317)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(977..1768,1769..1837))
                     /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     1600..1619
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1838..1942)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2010..2028
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2066..2088)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2188..2207
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2224..2679)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2361..2382)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2573..2592
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2754..2894
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2765..2894
                     /label=AAV2 ITR
     polyA_signal    2915..3139
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(join(3142..3855,3856..3858,3859..3861))
                     /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(3145..3855,3856..3858,3859..3861))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3187..3208)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3536..3555
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3795..3816
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3858..3867
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     LTR             3895..4163
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     promoter        complement(4176..4387)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_RNA        complement(4445..4520)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4542..4790)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4600..4619)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4770..4790)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(4820..5530)
                     /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(5098..5117)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5367..5385
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(5531..5587)
                     /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"
ORIGIN
        1 gtaccaagct tggcatcgat ggccgcagga acccctagtg atggagttgg ccactccctc
       61 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggcgg
      121 cctcagtgag cgagcgagcg cgcagctgcc tgcaggacat gtgagcaaaa ggccagcaaa
      181 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
      241 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
      301 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
      361 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
      421 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
      481 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
      541 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
      601 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
      661 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
      721 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
      781 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
      841 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
      901 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
      961 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     1021 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     1081 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     1141 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     1201 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     1261 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     1321 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     1381 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     1441 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     1501 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     1561 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     1621 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     1681 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     1741 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     1801 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     1861 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     1921 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     1981 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     2041 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2101 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2161 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     2221 ataaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     2281 cattttttaa ccaatagacc gaaatcggca aaatccctta taaatcaaaa gaatagcccg
     2341 agatagagtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2401 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2461 ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2521 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2581 aagcgaaagg agcgggcgct aaggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2641 ccacacccgc cgcgcttaat gcgccgctac agggcgcgta ctatggttgc tttgacgtat
     2701 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gcccctgcag
     2761 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac
     2821 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     2881 cactaggggt tcctgcggcc gacgcggccg caagccatag agcccaccgc atccccagca
     2941 tgcctgctat tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga
     3001 atagaatgac acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca
     3061 gtgggagtgg caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc
     3121 tggcaactag aaggcacagt cttacttgta cagctcgtcc atgccgagag tgatcccggc
     3181 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
     3241 ggcggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
     3301 ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat
     3361 cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga cgttgtggct
     3421 gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
     3481 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
     3541 gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc
     3601 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc
     3661 gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
     3721 gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa
     3781 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
     3841 ctcctcgccc ttgctcacca tggtggcacc ggtgtaggcg ccggtcacag cttggatctg
     3901 taacggcgca gaacagaaaa cgaaacaaag acgtagagtt gagcaagcag ggtcaggcaa
     3961 agcgtggaga gccggctgag tctaggtagg ctccaaggga gcgccggaca aaggcccggt
     4021 ctcgacctga gctttaaact tacctagacg gcggacgcag ttcaggaggc accacaggcg
     4081 ggaggcggca gaacgcgact caaccggcgt ggatggcggc ctcaggtagg gcggcgggcg
     4141 cgtgaaggag agatgcgagc ccctcgaagc ttcagctgtg ttctggcggc aaacccgttg
     4201 cgaaaaagaa cgttcacggc gactactgca cttatatacg gttctccccc accctcggga
     4261 aaaaggcgga gccagtacac gacatcactt tcccagttta ccccgcgcca ccttctctag
     4321 gcacccgttc aattgccgac ccctcccccc aacttctcgg ggactgtggg cgatgtgcgc
     4381 tctgcccact gacgggcacc ggagcgatcg cagatccttg ctagcctagc tctaaaacaa
     4441 aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt
     4501 gctatttcta gctctaaaac caagtgcttc taggcggact cggtgtttcg tcctttccac
     4561 aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc
     4621 attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac
     4681 gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttccaattat ctctctaaca
     4741 gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc ctcgagccgc
     4801 aagtgcttct aggcggactt tacttgtaca gctcgtccat gccgccggtg gagtggcggc
     4861 cctcggcgcg ttcgtactgt tccacgatgg tgtagtcctc gttgtgggag gtgatgtcca
     4921 acttgatgtt gacgttgtag gcgccgggca gctgcacggg cttcttggcc ttgtaggtgg
     4981 tcttgacctc agcgtcgtag tggccgccgt ccttcagctt cagcctctgc ttgatctcgc
     5041 ccttcagggc gccgtcctcg gggtacatcc gctcggagga ggcctcccag cccatggtct
     5101 tcttctgcat tacggggccg tcggagggga agttggtgcc gcgcagcttc accttgtaga
     5161 tgaactcgcc gtcctgcagg gaggagtcct gggtcacggt caccacgccg ccgtcctcga
     5221 agttcatcac gcgctcccac ttgaagccct cggggaagga cagcttcaag tagtcgggga
     5281 tgtcggcggg gtgcttcacg taggccttgg agccgtacat gaactgaggg gacaggatgt
     5341 cccaggcgaa gggcaggggg ccacccttgg tcaccttcag cttggcggtc tgggtgccct
     5401 cgtaggggcg gccctcgccc tcgccctcga tctcgaactc gtggccgttc acggagccct
     5461 ccatgtgcac cttgaagcgc atgaactcct tgatgatggc catgttatcc tcctcgccct
     5521 tgctcaccat tggcccggga ttctcttcga catcccctgc ttgtttcaac agggagaagt
     5581 tagtggcgcc gcaagtgctt ctaggcggac tg
//
