LOCUS       sequence-75952-e        6984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cetn1 genomic region was placed in pCAG-EGxxFP. Positive control for
            DSB mediated EGFP reconstitution. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6984)
  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 6984)
  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-Cetn1
FEATURES             Location/Qualifiers
     source          1..6984
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1741..1743,1744..1746,1747..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     3514..3535
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3664..3683)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3729..3784
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3783..3802)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4139..4158)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4140..4158)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4168..4185)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4168..4184)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4177..4199)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4326..4781
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4413..4432)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4623..4644
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4807..4911
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4912..4980,4981..5772)
                     /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(5130..5149)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5943..6531
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6432..6451
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6685..6702
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6819..6840
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6855..6860,6861..6878,6879..6885)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6893..6909
                     /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     6898..6920
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6917..6933
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6917..6933
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6952..6972
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6954..6972
                     /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 tgaggatcca gtcacttaat aaaggttggt ctttttcatg atcttaagaa actcttcctc
     2401 gttcacttct ccgtcgccat cacgatcagc ttcatcaatc atttcctgca gctcctcgtc
     2461 tgtgaggctt tcccccagct cattggccac acgcttgagg tttttgaatg aaattttccc
     2521 agtttcgtcg tcatcaaaca acctgaaagc cttcaggatt tcctctttgg tatctttctc
     2581 ggccatcttc tgagtcatca cagccaagaa gtcattgaag ctgatctttc ctgtggcctc
     2641 tttgtctact tctgaaatca ttttcttcat ctcttccttc ctgggttcaa agccaagtgc
     2701 tctcatggcc accttcagct ccttcacatc gatggtcccg ctcccatcag aatcgaagag
     2761 gtcaaaggct tcccgaactt cttgcttttg atcttcggtg agttcaggct taggacccac
     2821 ctttctcttg tagctggtag aggcaacgtt tgacttcctg aaggtggacg ccatcctcca
     2881 caacagttgg gatcttaacg gtgccctgac atcgcacgtg tgtgaggtgc cctgctagtg
     2941 cgcctgcgcc agcccctatg cgcctgcaga attcgatatc ggcaagctga ccctgaagtt
     3001 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     3061 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     3121 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     3181 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     3241 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     3301 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     3361 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     3421 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     3481 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     3541 cgccgggatc actctcggca tggacgagct gtacaagtaa ctcgagactc ctcaggtgca
     3601 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     3661 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     3721 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     3781 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     3841 ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag
     3901 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
     3961 gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct
     4021 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
     4081 catagctgtc cctcttctct tatgaagatc cctcgactta attaaggtac ccaattcgcc
     4141 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     4201 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     4261 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     4321 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4381 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4441 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4501 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     4561 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4621 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     4681 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4741 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     4801 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4861 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4921 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4981 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5041 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5101 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5161 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     5221 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     5281 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     5341 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     5401 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     5461 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     5521 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     5581 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     5641 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5701 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5761 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5821 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5881 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5941 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6001 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6061 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     6121 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6181 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     6241 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     6301 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     6361 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     6421 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     6481 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6541 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6601 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6661 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     6721 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     6781 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     6841 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     6901 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
     6961 cctcactaaa gggaacaaaa gctg
//
