LOCUS       sequence-178918-        4722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MMEJ donor for fusing a p2A-mCherry reporter to mouse Actb. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4722)
  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 4722)
  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 MMEJ donor
FEATURES             Location/Qualifiers
     source          1..4722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             306..362
                     /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             363..1073
                     /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(508..526)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     776..795
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1155..1174)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1156..1174)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1181..1198)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1181..1197)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1190..1212)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1338..1766
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1425..1444)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1635..1656
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             2110..2904
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2164..2183)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2774..2793
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2922..2990,2991..3782)
                     /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(3140..3159)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3953..4541
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4442..4461
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4695..4712
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gtaccagtcc gcctagaagc acttgcggcc cctccatcgt gcaccgcaag tgcttcttaa
      301 ttaacgccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag aatcccgggc
      361 caatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
      421 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
      481 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
      541 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
      601 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
      661 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
      721 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
      781 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
      841 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
      901 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
      961 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     1021 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taaggcgcgc
     1081 cgcggactgt tactgagctg cccgcaagtg cttctaggcg gactctcgag catgcatcta
     1141 gagggcccaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac
     1201 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     1261 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     1321 gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1381 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1441 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1501 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1561 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1621 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1681 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1741 gatttaacaa aaatttaacg cgaattttaa caaaattcag ggcgcaaggg ctgctaaagg
     1801 aagcggaaca cgtagaaagc cagtccgcag aaacggtgct gaccccggat gaatgtcagc
     1861 tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt
     1921 gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga accggaattg
     1981 ccagctgggg cgccctctgg taaggttggg aagccctgca aagtaaactg gatggctttc
     2041 ttgccgccaa ggatctgatg gcgcagggga tcaagatctg atcaagagac aggatgagga
     2101 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     2161 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     2221 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     2281 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     2341 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     2401 ccggggcagg atctcctgtc atcccacctt gctcctgccg agaaagtatc catcatggct
     2461 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     2521 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     2581 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     2641 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     2701 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     2761 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     2821 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     2881 cgccttcttg acgagttctt ctgaattgaa aaaggaagag tatgagtatt caacatttcc
     2941 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3001 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     3061 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     3121 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     3181 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     3241 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     3301 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     3361 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     3421 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     3481 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     3541 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     3601 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     3661 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     3721 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3781 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     3841 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3901 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3961 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4021 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4081 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4141 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4201 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4261 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4321 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4381 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4441 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4501 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4561 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4621 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4681 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga ag
//
