LOCUS       sequence-243398-        6702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Cre enzyme in mammalian cells with floxed genes to achieve 
            somatic knockout. GFP is simultaneously expressed for convenient 
            tracking the transfected cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6702)
  AUTHORS   Geng A, Qiu R, Murai K, Liu J, Wu X, Zhang H, Farhoodi H, Duong N, 
            Jiang M, Yee JK, Tsark W, Lu Q
  TITLE     KIF20A/MKLP2 regulates the division modes of neural progenitor cells
            during cortical development.
  JOURNAL   Nat Commun. 2018 Jul 13;9(1):2707. doi: 10.1038/s41467-018-05152-1.
  PUBMED    30006548
REFERENCE   2  (bases 1 to 6702)
  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: "Nat 
            Commun."; date: "2018-07-13"; volume: "9(1):2707. doi"; pages: " 
            10.1038/s41467-018-05152-1"
COMMENT     Sequence Label: CAG-Cremyc-2A-GFP
FEATURES             Location/Qualifiers
     source          1..6702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        119..498
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        500..777
                     /label=chicken beta-actin promoter
     intron          778..1794
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1718..1740
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1802..1821
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1904..2932
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             2933..2962
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2978..3034
                     /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"
     CDS             join(3035..3037,3038..3040,3041..3754)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3035..3037,3038..3040,3041..3751)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3341..3360)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3688..3709
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3838..3857)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3903..3958
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3957..3976)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4319..4335)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4319..4335)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4332..4354)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4343..4359
                     /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(4367..4373,4374..4391,4392..4397))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4412..4433
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4492..4687
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4538..4673
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4693..4827
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4730..4749)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4784..4803
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4895..4912)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5066..5654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5146..5165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5825..6616,6617..6685))
                     /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     6448..6467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6686..6702,1..88))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
       61 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgggtcga
      121 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      181 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      241 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      301 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      361 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      421 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      481 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
      541 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
      601 ggggcggggg gggggggggg gcgcgcgccr ggsggggsgg ggsggggsgr ggggsggggs
      661 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      721 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      781 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      841 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      901 ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc
      961 cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg
     1021 tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc
     1081 gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc
     1141 ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc
     1201 gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc
     1261 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1321 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1381 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1441 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1501 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1561 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1621 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1681 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1741 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1801 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaatttcg aggggcagag
     1861 ccgatcctgt acactttact taaaaccatt atctgagtgt gaaatgtcca atttactgac
     1921 cgtacaccaa aatttgcctg cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa
     1981 cctgatggac atgttcaggg atcgccaggc gttttctgag catacctgga aaatgcttct
     2041 gtccgtttgc cggtcgtggg cggcatggtg caagttgaat aaccggaaat ggtttcccgc
     2101 agaacctgaa gatgttcgcg attatcttct atatcttcag gcgcgcggtc tggcagtaaa
     2161 aactatccag caacatttgg gccagctaaa catgcttcat cgtcggtccg ggctgccacg
     2221 accaagtgac agcaatgctg tttcactggt tatgcggcgg atccgaaaag aaaacgttga
     2281 tgccggtgaa cgtgcaaaac aggctctagc gttcgaacgc actgatttcg accaggttcg
     2341 ttcactcatg gaaaatagcg atcgctgcca ggatatacgt aatctggcat ttctggggat
     2401 tgcttataac accctgttac gtatagccga aattgccagg atcagggtta aagatatctc
     2461 acgtactgac ggtgggagaa tgttaatcca tattggcaga acgaaaacgc tggttagcac
     2521 cgcaggtgta gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc gatggatttc
     2581 cgtctctggt gtagctgatg atccgaataa ctacctgttt tgccgggtca gaaaaaatgg
     2641 tgttgccgcg ccatctgcca ccagccagct atcaactcgc gccctggaag ggatttttga
     2701 agcaactcat cgattgattt acggcgctaa ggatgactct ggtcagagat acctggcctg
     2761 gtctggacac agtgcccgtg tcggagccgc gcgagatatg gcccgcgctg gagtttcaat
     2821 accggagatc atgcaagctg gtggctggac caatgtaaat attgtcatga actatatccg
     2881 taacctggat agtgaaacag gggcaatggt gcgcctgctg gaagatggcg atgaacaaaa
     2941 actcatctca gaagaggatc tggaattcgg atccggagcc acgaacttct ctctgttaaa
     3001 gcaagcagga gacgtggaag aaaaccccgg tcctatggtg agcaagggcg aggagctgtt
     3061 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     3121 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     3181 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     3241 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     3301 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     3361 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     3421 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     3481 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     3541 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     3601 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     3661 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     3721 gatcactctc ggcatggacg agctgtacaa gtaagaattc actcctcagg tgcaggctgc
     3781 ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct
     3841 ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg
     3901 ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact
     3961 cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag
     4021 tttggcaaca tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca
     4081 tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg
     4141 aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt
     4201 ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc
     4261 tgtccctctt ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt
     4321 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4381 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4441 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa
     4501 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4561 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4621 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4681 ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4741 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4801 atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga
     4861 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4921 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4981 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5041 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5101 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5161 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5221 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5281 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5341 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5401 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5461 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5521 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5581 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5641 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5701 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5761 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5821 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5881 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5941 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6001 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6061 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6121 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6181 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6241 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6301 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6361 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6421 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6481 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6541 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6601 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6661 cgacacggaa atgttgaata ctcatactct tcctttttca at
//
