LOCUS       sequence-182594-        4658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NHEJ 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 4658)
  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 4658)
  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
FEATURES             Location/Qualifiers
     source          1..4658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(94..115)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     306..325
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     538..560
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     552..569
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     553..569
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     576..595
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        576..594
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(649..1359)
                     /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(927..946)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1196..1214
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1360..1416)
                     /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"
     primer_bind     complement(1465..1481)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1465..1481)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1478..1500)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1489..1505
                     /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)."
     promoter        complement(join(1513..1519,1520..1537,1538..1543))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1558..1579
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1696..1713)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1867..2455)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1947..1966)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2626..3417,3418..3486))
                     /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     3249..3268
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(3504..4298)
                     /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(3615..3634)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4225..4244
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(join(4642..4658,1..412))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
       61 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
      121 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
      181 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
      241 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
      301 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
      361 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc
      421 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
      481 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
      541 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat
      601 tgggccctct agatgcatgc tcgagccgca agtgcttcta ggcggacttt acttgtacag
      661 ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt tcgtactgtt ccacgatggt
      721 gtagtcctcg ttgtgggagg tgatgtccaa cttgatgttg acgttgtagg cgccgggcag
      781 ctgcacgggc ttcttggcct tgtaggtggt cttgacctca gcgtcgtagt ggccgccgtc
      841 cttcagcttc agcctctgct tgatctcgcc cttcagggcg ccgtcctcgg ggtacatccg
      901 ctcggaggag gcctcccagc ccatggtctt cttctgcatt acggggccgt cggaggggaa
      961 gttggtgccg cgcagcttca ccttgtagat gaactcgccg tcctgcaggg aggagtcctg
     1021 ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg cgctcccact tgaagccctc
     1081 ggggaaggac agcttcaagt agtcggggat gtcggcgggg tgcttcacgt aggccttgga
     1141 gccgtacatg aactgagggg acaggatgtc ccaggcgaag ggcagggggc cacccttggt
     1201 caccttcagc ttggcggtct gggtgccctc gtaggggcgg ccctcgccct cgccctcgat
     1261 ctcgaactcg tggccgttca cggagccctc catgtgcacc ttgaagcgca tgaactcctt
     1321 gatgatggcc atgttatcct cctcgccctt gctcaccatt ggcccgggat tctcttcgac
     1381 atcccctgct tgtttcaaca gggagaagtt agtggcgccg caagtgcttc taggcggact
     1441 ggtaccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     1501 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     1561 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     1621 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     1681 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     1741 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     1801 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     1861 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     1921 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     1981 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2041 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2101 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2161 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2221 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2281 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2341 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2401 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2461 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2521 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     2581 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     2641 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     2701 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     2761 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     2821 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     2881 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     2941 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3001 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3061 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3121 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3181 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3241 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     3301 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     3361 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     3421 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     3481 actcatactc ttcctttttc aattcagaag aactcgtcaa gaaggcgata gaaggcgatg
     3541 cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg
     3601 ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
     3661 cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc
     3721 aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc
     3781 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg
     3841 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
     3901 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
     3961 actttctcgg caggagcaag gtgggatgac aggagatcct gccccggcac ttcgcccaat
     4021 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc
     4081 gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac
     4141 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
     4201 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
     4261 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc
     4321 tcttgatcag atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag
     4381 tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg
     4441 cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc
     4501 tgctttctct ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc
     4561 cggggtcagc accgtttctg cggactggct ttctacgtgt tccgcttcct ttagcagccc
     4621 ttgcgccctg aattttgtta aaattcgcgt taaatttt
//
