LOCUS       sequence-201896-        8458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 coding gene template with optimized sequence for human codon 
            usage, also expresses EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8458)
  AUTHORS   Cho N, Seo HN, Ryu T, Kwon E, Huh S, Noh J, Yeom H, Hwang B, Ha H, 
            Lee JH, Kwon S, Bang D
  TITLE     High-throughput construction of multiple cas9 gene variants via 
            assembly of high-depth tiled and sequence-verified oligonucleotides.
  JOURNAL   Nucleic Acids Res. 2018 May 18;46(9):e55. doi: 10.1093/nar/gky112.
  PUBMED    29529247
REFERENCE   2  (bases 1 to 8458)
  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: "Nucleic 
            Acids Res."; date: "2018-05-18"; volume: "46(9):e55. doi"; pages: " 
            10.1093/nar/gky112"
COMMENT     Sequence Label: pCAG-B2UP49_GFP
FEATURES             Location/Qualifiers
     source          1..8458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1071..1093
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1155..1174
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             4529..4549
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4565..4618
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(4619..4621,4622..4624,4625..5338)
                     /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(4619..4621,4622..4624,4625..5335)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4664..4685)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4925..4944)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5272..5293
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5381..5502
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5418..5437)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5472..5491
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5551..5567)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5551..5567)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5564..5586)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5575..5591
                     /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(5599..5605,5606..5623,5624..5629))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5644..5665
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5782..5799)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5953..6541)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6033..6052)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6712..7503,7504..7572))
                     /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     7335..7354
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7573..7677)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7756..7775)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7947..8326
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
       61 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
      121 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
      181 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
      241 gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc
      301 gggagggccc tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg
      361 ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg
      421 ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt
      481 gcgggggggg ttgtcgggca gcagcacggg gccgtcgccg atggggaaca aaggctgcgt
      541 gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc
      601 ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg
      661 tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc
      721 gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg
      781 gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg
      841 cgagagggcg cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg
      901 ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat
      961 gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg
     1021 gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt
     1081 ctggcgtgtg accggcggct ctagcgcctc tgctaaccat gttcatgcct tcttcttttt
     1141 cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt
     1201 cgcggccgcc accatgtcaa ggagcctcac tttcagcttc gacatcggtt acgcttccat
     1261 tgggtgggcc gtgattgcta gtgcctccca tgacgacgcc gatccttctg tgtgtgggtg
     1321 cggaacggtg ctgtttccaa aggatgactg tcaggcattc aaaaggagag agtatagaag
     1381 gctgcgcagg aacatccgaa gtcgcagagt ccggatcgag cggatcggga gactccttgt
     1441 gcaagctcag atcatcacac ccgaaatgaa agaaacctcc ggccacccag caccttttta
     1501 ccttgcaagc gaggccctga aagggcatcg gacactggct ccaattgaat tgtggcacgt
     1561 gcttaggtgg tatgcacaca accgagggta tgacaacaac gcgtcttggt ctaactctct
     1621 ctcagaagac ggaggtaacg gggaagatac tgagcgcgtc aaacacgctc aggatctgat
     1681 ggacaagcac ggaaccgcaa ccatggccga aaccatctgt cgggagctta agctggagga
     1741 gggaaaggcg gatgccccca tggaggtgag tacgccggct tacaaaaatc tgaatactgc
     1801 gtttcccagg ctgatcgttg agaaggaggt gagacggatt ctcgagctga gtgcgcccct
     1861 gatcccaggg ctgacagccg agatcattga gctgatcgcc caacaccatc ctttgacgac
     1921 agaacagcgg ggggtgctgc tgcagcacgg tatcaagctc gcacgaaggt atcgcggcag
     1981 tctccttttt ggccaactga taccacggtt cgacaaccga attatcagca gatgccctgt
     2041 aacatgggcg caggtctatg aggccgagct gaagaaaggg aatagcgagc agtccgctag
     2101 agaacgagct gaaaaattgt ctaaggtccc cacggccaat tgccctgaat tttacgagta
     2161 tcgcatggcc cgcattctgt gcaacatccg ggccgacggt gaacctctct ctgcggaaat
     2221 cagaagggag ctgatgaatc aggcacgcca agaggggaaa ctgaccaagg cctccctgga
     2281 gaaagccata agttctagac tggggaagga aactgaaacc aacgtcagca attacttcac
     2341 ccttcatccc gactccgaag aggcactgta cttgaatccc gccgttgagg tcctgcagcg
     2401 ctcagggatt gggcagatac tttcacccag cgtgtacaga attgctgcca atcgcctgag
     2461 acgaggaaaa agtgtgacac ccaattacct gctcaacctc ctgaagtcca ggggtgagtc
     2521 aggagaggcc ctggaaaaga agattgagaa agagagtaag aagaaagagg cagattatgc
     2581 cgacactcct ctgaaaccta agtacgccac cggccgcgcc ccgtatgcac gcaccgttct
     2641 caagaaggtg gtcgaggaaa tactggatgg ggaggatcct acccggccag ccaggggcga
     2701 ggctcaccct gacggggaat tgaaagccca cgatgggtgt ctgtattgtc ttcttgatac
     2761 cgactccagc gtaaatcagc accagaagga gcggcggctg gataccatga ctaataacca
     2821 tctcgtgaga cacaggatgc tcatcctcga cagactgctc aaagacctca ttcaggactt
     2881 tgccgatgga caaaaggaca ggatctccag ggtgtgcgtc gaagtcggca aagaactgac
     2941 aacattttca gccatggact ctaagaagat tcagagagag ctcacgctta ggcagaagtc
     3001 acacactgac gctgtcaata gactcaagag aaagctgcct ggaaaagccc ttagcgcaaa
     3061 tctgataagg aagtgccgga tcgccatgga catgaactgg acctgcccct ttacgggtgc
     3121 gacgtacgga gatcatgagc tggagaacct ggaactggag cacatcgttc ctcacagctt
     3181 tcgccaatct aacgcgctgt catccctggt gctcacctgg cctggcgtca atcgcatgaa
     3241 ggggcaacgg acaggatacg acttcgttga gcaggaacag gaaaatcccg tgccagataa
     3301 gcccaacctg cacatttgtt ccttgaataa ctatcgcgaa ctggtggaga aactcgacga
     3361 taagaaaggg catgaggacg acagaaggcg caagaagaaa cggaaggccc ttcttatggt
     3421 gagaggattg agccacaagc atcagtcaca gaatcatgag gctatgaagg agattggaat
     3481 gaccgagggc atgatgacac agtcctcaca tcttatgaag ctcgcttgta aatcaatcaa
     3541 gacatcactg cccgacgcac atatcgatat gatccccggc gccgttaccg ccgaggtgag
     3601 aaaggcctgg gatgtattcg gagttttcaa ggagctctgt cccgaggccg cagatcctga
     3661 tagcgggaag atcctcaagg aaaacctgcg ctcacttacg catctgcatc acgcacttga
     3721 tgcctgcgta ctggggctga tcccatatat tatccctgct catcataatg gtcttctgcg
     3781 gcgagtgctg gctatgcgac gcatcccaga gaagctgata ccccaggtgc ggcctgtcgc
     3841 aaatcagcgg cattacgtgt tgaatgacga tggacgaatg atgctgaggg acctcagcgc
     3901 ctccctgaag gagaacatcc gcgagcagct gatggagcaa cgggtgatcc agcatgtgcc
     3961 ggcagacatg ggcggagccc tgctgaaaga aactatgcaa agagtcttgt ctgtggatgg
     4021 atctggagaa gacgcaatgg taagtctgtc caagaagaag gacggcaaaa aggagaagaa
     4081 ccaggtgaag gcctccaaat tggtcggggt gttccccgaa gggccatcaa aacttaaggc
     4141 acttaaggct gccatcgaga tcgacggcaa ctacggagtg gcattggatc cgaaacctgt
     4201 cgtgatcaga cacatcaaag tgtttaaaag aatcatggcg ctgaaggagc agaacggtgg
     4261 taagccggtt aggatcctga agaagggaat gttgattcac ctgacctcct caaaagatcc
     4321 taagcacgcc ggggtctgga gaattgagtc tatccaggat tcaaaggggg gggtcaagct
     4381 cgacctgcag agggctcatt gcgccgtgcc aaaaaataag acacacgaat gcaattggag
     4441 ggaggtcgac ctgatctcac tgctcaaaaa gtaccagatg aagaggtacc caacttcata
     4501 tacgggcacc ccaagaagca gggctgaccc caagaagaag aggaaggtga ggtccggcgg
     4561 cggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggcccaat
     4621 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     4681 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     4741 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     4801 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     4861 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     4921 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     4981 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     5041 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     5101 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     5161 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     5221 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     5281 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaact
     5341 gcagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5401 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5461 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5521 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5581 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctagggtgc
     5641 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5701 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5761 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5821 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5881 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5941 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6001 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6061 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6121 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6181 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6241 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6301 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6361 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6421 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6481 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6541 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6601 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6661 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6721 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6781 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6841 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6901 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6961 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7021 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7081 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7141 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7201 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7261 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7321 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7381 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7441 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7501 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7561 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7621 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7681 atttccccga aaagtgccac ctgacgtcga cggatcggga gatcgatctc ccgatcccct
     7741 agggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     7801 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     7861 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     7921 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
     7981 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     8041 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     8101 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     8161 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     8221 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     8281 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
     8341 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
     8401 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggg cgcgcgcc
//
