LOCUS       sequence-306370-        7224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to detect crossover-type DNA recombination events elicited by 
            iso-positional nicking of two homologous DNA fragments 
            (recombination analogous to Holliday's model).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7224)
  AUTHORS   Hyodo T, Rahman ML, Karnan S, Ito T, Toyoda A, Ota A, Wahiduzzaman 
            M, Tsuzuki S, Okada Y, Hosokawa Y, Konishi H
  TITLE     Tandem Paired Nicking Promotes Precise Genome Editing with Scarce 
            Interference by p53.
  JOURNAL   Cell Rep. 2020 Jan 28;30(4):1195-1207.e7. doi: 
            10.1016/j.celrep.2019.12.064.
  PUBMED    31995758
REFERENCE   2  (bases 1 to 7224)
  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"; doi: 
            "10.1016/j.celrep.2019.12"; journalName: "Cell Rep"; date: 
            "2020-01-28- 28"; volume: "30"; issue: "4"; pages: "1195-1207.e7"
COMMENT     Sequence Label: prCO
FEATURES             Location/Qualifiers
     source          1..7224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             8..477
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    542..741
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             942..1358
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1288..1310
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1326..1342
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1423..1443
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1465..1485
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1699..1708
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1705..1707,1708..1710,1711..2169)
                     /codon_start=1
                     /product="improved N-terminal fragment of mVenus for use in
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN155(I152L)
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA"
     primer_bind     complement(1750..1771)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     intron          2278..2507
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2490..2509
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2636..3098
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2692..2709)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2919..2938
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3112..3906
                     /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="MAEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3166..3185)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3776..3795
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4484..4505
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4718..5187
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5317..5334)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5488..6076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5568..5587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6236..7027,7028..7096))
                     /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     6859..6878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7097..7201)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
       61 gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt
      121 tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag
      181 tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc
      241 ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga
      301 gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc
      361 gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc
      421 tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt
      481 gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg
      541 caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta
      601 tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
      661 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
      721 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
      781 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
      841 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt
      901 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc
      961 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     1021 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     1081 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     1141 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     1201 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     1261 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     1321 cctcccttta tccagccctc actccttctc taggcgccgg ccggatccca gtgtggtggt
     1381 acgtaggaat tcagatccgc cgccactatg ggatcaagat cgccaaaaaa gaagagaaag
     1441 gtgccgaaga agcatgcagc accaccaaaa aaaaaacgaa aagtagaaga cccacgaggc
     1501 aacaccagcg gcgtgctgag cacccccaag gccaagaggg ccaagcaccc ccccggcacc
     1561 gagaagccca ggagcaggag ccagagcgag cagcccgcca cctgccccat ctgctacgcc
     1621 gtgatcaggc agagcaggaa cctgaggagg cacctggagc tgaggcactt cgccaagccc
     1681 ggcgtggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     1741 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1801 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1861 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1921 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1981 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     2041 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2101 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     2161 atcatggccg acaagcagaa gaacggcatc aggtgaactt caataaaagc tggccgcata
     2221 gataactgat ccagtgtgct ggaattaatt cgctgtctgc gagggccagc tgttggggtg
     2281 agtactccct ctcaaaagcg ggcatgactt ctgcgctaag attgtcagtt tccaaaaacg
     2341 aggaggattt gatattcacc tggcccgcgg tgatgccttt gagggtggcc gcgtccatct
     2401 ggtcagaaaa gacaatcttt ttgttgtcaa gcttgaggtg tggcaggctt gagatctggc
     2461 catacacttg agtgacaatg acatccactt tgcctttctc tccacaggtg tccactccca
     2521 ggtccaactg caggtcgagc gtcgagaacg ttactggccg aagccgcttg gaataaggcc
     2581 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     2641 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     2701 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     2761 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     2821 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     2881 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     2941 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     3001 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     3061 acggggacgt ggttttcctt tgaaaaacac gatgataagc ttgccacaac catggctgaa
     3121 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3181 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3241 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     3301 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3361 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3421 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3481 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3541 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3601 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgatgat
     3661 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3721 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3781 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3841 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3901 ttctgagggg atcaattctc tagagctagg gataacaggg taatacctac ggcaagctga
     3961 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     4021 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     4081 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     4141 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     4201 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     4261 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     4321 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     4381 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     4441 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     4501 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa atcgataaaa
     4561 taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt
     4621 ggcaagctag cttaagtaac gccattttgc aaggcatgga aaaatacata actgagaata
     4681 gagaagttca gatcaaggtc aggaacagat ggaacagctg aatatgggcc aaacaggata
     4741 tctgtggtaa gcagttcctg ccccggctca gggccaagaa cagatggaac agctgaatat
     4801 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
     4861 ggtccccaga tgcggtccag ccctcagcag tttctagaga accatcagat gtttccaggg
     4921 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct
     4981 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact
     5041 cggggcgcca gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc aataaaccct
     5101 cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
     5161 tgactacccg tcagcggggg tctttcacat gcagcatgta tcaaaattaa tttggttttt
     5221 tttcttaagt atttacatta aatggccata gttgcattaa tgaatcggcc aacgcgcggg
     5281 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5341 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5401 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5461 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5521 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5581 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5641 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5701 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5761 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5821 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5881 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5941 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6001 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6061 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6121 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6181 ccttttgcgg ccgcaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6241 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6301 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6361 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6421 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6481 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6541 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6601 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6661 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6721 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6781 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6841 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6901 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6961 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7021 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7081 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7141 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7201 gcacatttcc ccgaaaagtg ccac
//
