LOCUS       sequence-178906-        5623 bp    DNA     circular SYN 15-DEC-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 5623)
  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 5623)
  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 NHEJ donor_U6_sgRNA_EF1a_GFP_polyA
FEATURES             Location/Qualifiers
     source          1..5623
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     CDS             193..249
                     /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             250..960
                     /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(395..413)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     663..682
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        990..1238
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     990..1010
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1161..1180
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1260..1335
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1393..1604
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1617..1885
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      1913..1922
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1919..1921,1922..1924,1925..2638)
                     /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(1919..1921,1922..1924,1925..2635)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1964..1985)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2225..2244)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2572..2593
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2641..2865
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2886..3026
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2886..3015
                     /label=AAV2 ITR
     rep_origin      3101..3556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3188..3207)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3398..3419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3573..3592)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3692..3714
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3752..3770)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3838..3942
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3943..4011,4012..4803)
                     /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(4161..4180)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4974..5562
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5463..5482
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccatc gatgccaagc ttggtaccag tccgcctaga
      181 agcacttgcg gcgccactaa cttctccctg ttgaaacaag caggggatgt cgaagagaat
      241 cccgggccaa tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
      301 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
      361 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
      421 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
      481 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
      541 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
      601 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
      661 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
      721 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
      781 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
      841 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
      901 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtaa
      961 agtccgccta gaagcacttg cggctcgagg agggcctatt tcccatgatt ccttcatatt
     1021 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     1081 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     1141 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     1201 tcttggcttt atatatcttg tggaaaggac gaaacaccga gtccgcctag aagcacttgg
     1261 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     1321 gcaccgagtc ggtgcttttt tgttttagag ctaggctagc aaggatctgc gatcgctccg
     1381 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
     1441 tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
     1501 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
     1561 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acagctgaag cttcgagggg
     1621 ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg ccgccatcca cgccggttga
     1681 gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac tgcgtccgcc gtctaggtaa
     1741 gtttaaagct caggtcgaga ccgggccttt gtccggcgct cccttggagc ctacctagac
     1801 tcagccggct ctccacgctt tgcctgaccc tgcttgctca actctacgtc tttgtttcgt
     1861 tttctgttct gcgccgttac agatccaagc tgtgaccggc gcctacaccg gtgccaccat
     1921 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1981 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     2041 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     2101 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     2161 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     2221 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     2281 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2341 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2401 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2461 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2521 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2581 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaga
     2641 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     2701 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     2761 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     2821 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttgc ggccgcgtcg
     2881 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     2941 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3001 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     3061 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     3121 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     3181 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3241 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3301 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     3361 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3421 acaacactca accctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     3481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3541 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3601 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3661 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3721 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     3781 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     3841 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3901 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3961 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4021 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4081 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4141 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4201 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4261 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4321 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4381 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4441 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4501 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4561 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4621 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4681 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4741 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4801 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4861 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4921 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4981 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5041 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5101 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     5161 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5221 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5281 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5341 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5401 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5461 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5521 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5581 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
