LOCUS       sequence-220772-        6699 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 6699)
  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 6699)
  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..6699
                     /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..3751)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3035..3037,3038..3748)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3338..3357)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3685..3706
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3835..3854)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3900..3955
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3954..3973)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4316..4332)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4316..4332)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4329..4351)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4340..4356
                     /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(4364..4370,4371..4388,4389..4394))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4409..4430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4489..4684
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4535..4670
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4690..4824
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4727..4746)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4781..4800
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4892..4909)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5063..5651)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5143..5162)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5822..6613,6614..6682))
                     /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     6445..6464
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6683..6699,1..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 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc
      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 tcctgtgagc aagggcgagg agctgttcac
     3061 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     3121 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     3181 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     3241 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     3301 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     3361 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     3421 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     3481 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     3541 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     3601 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     3661 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     3721 cactctcggc atggacgagc tgtacaagta agaattcact cctcaggtgc aggctgccta
     3781 tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact gagatctttt
     3841 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta
     3901 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg
     3961 aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt
     4021 ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca
     4081 gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg
     4141 ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc
     4201 cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt
     4261 ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat
     4321 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4381 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4441 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta
     4501 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4561 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4621 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4681 caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4741 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4801 aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag
     4861 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4921 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4981 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5041 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5101 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5161 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5221 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5281 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5341 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5401 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5461 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5521 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5581 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5641 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5701 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5761 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5821 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5881 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5941 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6001 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6061 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6121 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6181 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6241 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6301 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6361 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6421 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6481 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6541 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6601 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6661 cacggaaatg ttgaatactc atactcttcc tttttcaat
//
