LOCUS Exported 6699 bp ds-DNA circular SYN 12-MAY-2021 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 CAG-Cremyc-2A-GFP 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6699 /organism="synthetic DNA construct" /mol_type="other DNA" 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 3035..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 3035..3748 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(3077..3098) /label=EGFP-N /note="EGFP, reverse primer" 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(4364..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" primer_bind 4597..4616 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" 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(5822..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(6683..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 //