LOCUS       sequence-447281-        6396 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  5' and 3' EGFP fragments that shares 482bp were placed under 
            ubiquitous CAG promoter. Used for validation of gRNA sequences by 
            DSB mediated EGFP reconstitution. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6396)
  AUTHORS   Mashiko D, Fujihara Y, Satouh Y, Miyata H, Isotani A, Ikawa M
  TITLE     Generation of mutant mice by pronuclear injection of circular 
            plasmid expressing Cas9 and single guided RNA.
  JOURNAL   Sci Rep. 2013 Nov 27;3:3355. doi: 10.1038/srep03355.
  PUBMED    24284873
REFERENCE   2  (bases 1 to 6396)
  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: "Sci Rep."; 
            date: "2013-11-27"; volume: "3:3355. doi"; pages: " 
            10.1038/srep03355"
COMMENT     Sequence Label: pCAG-EGxxFP
FEATURES             Location/Qualifiers
     source          1..6396
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..662
                     /label=chicken beta-actin promoter
     intron          663..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1603..1625
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1685..1704
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1732..1741
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1738..1740,1741..1743,1744..2256)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(1783..1804)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2910..2931
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3060..3079)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3125..3180
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3179..3198)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3535..3554)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3536..3554)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3564..3581)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3564..3580)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3573..3595)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3722..4177
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3809..3828)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4019..4040
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4203..4307
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4308..4376,4377..5168)
                     /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(4526..4545)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5339..5927
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5828..5847
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6081..6098
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6215..6236
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6251..6256,6257..6274,6275..6281)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6289..6305
                     /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     6294..6316
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6313..6329
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6313..6329
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6348..6368
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6350..6368
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 ccccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
      481 atgggggcgg gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc
      541 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
      601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      661 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      841 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1021 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1081 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1141 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1201 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     1261 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     1321 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     1381 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     1441 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     1501 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1561 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1621 tgaccggcgg ctctagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1681 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaatctag agccgccatg
     1741 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1801 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1861 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1921 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     1981 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2041 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2101 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2161 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2221 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2281 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactga
     2341 ggatccgcta gcctgcaggt cgacgaattc gatatcggca agctgaccct gaagttcatc
     2401 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     2461 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2521 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2581 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2641 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     2701 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     2761 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     2821 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     2881 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2941 gggatcactc tcggcatgga cgagctgtac aagtaactcg agactcctca ggtgcaggct
     3001 gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata ccactgagat
     3061 ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     3121 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     3181 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     3241 agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt
     3301 catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact
     3361 tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa
     3421 ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata
     3481 gctgtccctc ttctcttatg aagatccctc gacttaatta aggtacccaa ttcgccctat
     3541 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     3601 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     3661 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     3721 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3781 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3841 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3901 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3961 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4021 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4081 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4141 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     4201 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4261 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4321 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4381 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4441 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4501 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4561 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4621 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4681 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4741 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4801 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4861 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4921 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4981 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5041 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5101 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5161 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5221 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5281 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5341 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5401 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5461 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5521 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5581 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5641 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5701 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5761 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5821 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5881 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5941 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     6001 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     6061 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     6121 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     6181 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     6241 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     6301 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
     6361 actaaaggga acaaaagctg gagctcatcg atactc
//
