LOCUS       sequence-197922-        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"
     primer_bind     32..51
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     285..302
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    419..440
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(455..460,461..478,479..485)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    493..509
                     /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     498..520
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     517..533
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     517..533
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             618..674
                     /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             675..1385
                     /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(820..838)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1088..1107
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1467..1486)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1468..1486)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1493..1510)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1493..1509)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1502..1524)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1650..2078
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1737..1756)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1947..1968
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             2422..3216
                     /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(2476..2495)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3086..3105
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(3234..3302,3303..4094)
                     /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(3452..3471)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4265..4722,1..131)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
       61 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      121 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      181 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
      241 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
      301 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
      361 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
      421 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      481 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
      541 acgccaagct tggtaccagt ccgcctagaa gcacttgcgg cccctccatc gtgcaccgca
      601 agtgcttctt aattaacgcc actaacttct ccctgttgaa acaagcaggg gatgtcgaag
      661 agaatcccgg gccaatggtg agcaagggcg aggaggataa catggccatc atcaaggagt
      721 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
      781 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
      841 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
      901 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
      961 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     1021 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     1081 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt
     1141 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     1201 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     1261 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     1321 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     1381 agtaaggcgc gccgcggact gttactgagc tgcccgcaag tgcttctagg cggactctcg
     1441 agcatgcatc tagagggccc aattcgccct atagtgagtc gtattacaat tcactggccg
     1501 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     1561 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     1621 aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc
     1681 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1741 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1801 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1861 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1921 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1981 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2041 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaattc agggcgcaag
     2101 ggctgctaaa ggaagcggaa cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg
     2161 atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag
     2221 gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc
     2281 gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac
     2341 tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc tgatcaagag
     2401 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     2461 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     2521 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     2581 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     2641 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     2701 ttgggcgaag tgccggggca ggatctcctg tcatcccacc ttgctcctgc cgagaaagta
     2761 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     2821 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     2881 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     2941 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3001 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3061 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3121 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3181 atcgccttct atcgccttct tgacgagttc ttctgaattg aaaaaggaag agtatgagta
     3241 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     3301 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     3361 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     3421 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     3481 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     3541 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     3601 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     3661 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3721 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     3781 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3841 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3901 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3961 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4021 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4081 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4141 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4201 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4261 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4321 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4381 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     4441 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4501 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4561 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4621 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4681 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cg
//
