LOCUS       Exported                6383 bp ds-DNA     circular SYN 12-MAY-2021
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    pCAG-EGxxFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6383)
  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 6383)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6383
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        16..395
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        398..673
                     /label=chicken beta-actin promoter
     intron          674..1680
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1606..1628
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1688..1707
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1735..1744
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1741..2259
                     /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(1786..1807)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2913..2934
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3063..3082)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3128..3183
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3182..3201)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3538..3557)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3539..3557)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3567..3584)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3567..3583)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3576..3598)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3725..4180
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3812..3831)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4022..4043
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4206..4310
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4311..5171
                     /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(4529..4548)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5342..5930
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5831..5850
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6084..6101
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6218..6239
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6254..6284
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6292..6308
                     /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     6297..6319
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6316..6332
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6316..6332
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6351..6371
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6353..6371
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gagctcatcg atctcgacat tgattattga ctagttatta atagtaatca attacggggt
       61 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      121 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      181 taacgccaat agggactttc cattgacgtc aatgggtgga ctatttacgg taaactgccc
      241 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      301 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      361 agtacatcta cgtattagtc atcgctatta ccatgggtcg aggtgagccc cacgttctgc
      421 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      481 ttattttgtg cagcgatggg ggcggggggg gggggggcgc gcgccaggcg gggcggggcg
      541 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
      601 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
      661 cgcgcggcgg gcgggagtcg ctgcgttgcc ttcgccccgt gccccgctcc gcgccgcctc
      721 gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg
      781 cccttctcct ccgggctgta attagcgctt ggtttaatga cggctcgttt cttttctgtg
      841 gctgcgtgaa agccttaaag ggctccggga gggccctttg tgcggggggg agcggctcgg
      901 ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggcccgcgc tgcccggcgg
      961 ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc cgcgtgtgcg cgaggggagc
     1021 gcggccgggg gcggtgcccc gcggtgcggg ggggctgcga ggggaacaaa ggctgcgtgc
     1081 ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc ggcggtcggg ctgtaacccc
     1141 cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt
     1201 gcggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg
     1261 ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggccccgga
     1321 gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg
     1381 agagggcgca gggacttcct ttgtcccaaa tctggcggag ccgaaatctg ggaggcgccg
     1441 ccgcaccccc tctagcgggc gcgggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     1501 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccatctcca gcctcggggc
     1561 tgccgcaggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1621 gtgtgaccgg cggctctagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1681 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaatc tagagccgcc
     1741 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1801 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1861 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1921 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1981 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2041 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2101 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2161 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     2221 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     2281 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2341 tgaggatccg ctagcctgca ggtcgacgaa ttcgatatcg gcaagctgac cctgaagttc
     2401 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2461 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2521 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2581 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2641 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2701 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2761 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2821 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2881 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2941 gccgggatca ctctcggcat ggacgagctg tacaagtaac tcgagactcc tcaggtgcag
     3001 gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga
     3061 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     3121 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     3181 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     3241 tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga
     3301 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg
     3361 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta
     3421 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc
     3481 atagctgtcc ctcttctctt atgaagatcc ctcgacttaa ttaaggtacc caattcgccc
     3541 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     3601 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     3661 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     3721 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3781 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3841 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3901 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3961 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4021 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     4081 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     4141 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     4201 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4261 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4321 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4381 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4441 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4501 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4561 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4621 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4681 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4741 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4801 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4861 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4921 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4981 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5041 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5101 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5161 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5221 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5281 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5341 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5401 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5461 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     5521 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5581 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5641 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5701 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5761 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5821 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5881 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5941 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6001 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6061 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     6121 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     6181 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     6241 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     6301 gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc
     6361 ctcactaaag ggaacaaaag ctg
//
