LOCUS       sequence-209831-        8501 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 8501)
  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 8501)
  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..8501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        302..580
                     /label=chicken beta-actin promoter
     primer_bind     1546..1568
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1630..1649
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             5004..5024
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5040..5093
                     /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(5094..5096,5097..5099,5100..5813)
                     /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(5094..5096,5097..5099,5100..5810)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5139..5160)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5400..5419)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5747..5768
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5856..5977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5893..5912)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5947..5966
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6026..6042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6026..6042)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6039..6061)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6050..6066
                     /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(6074..6080,6081..6098,6099..6104))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6119..6140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6257..6274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6428..7016)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6508..6527)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7187..7978,7979..8047))
                     /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     7810..7829
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8048..8152)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8231..8250)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8422..8501,1..300)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
       61 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      121 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      181 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      241 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      301 gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca
      361 attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg
      421 ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg
      481 tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg
      541 gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct
      601 tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
      661 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg
      721 gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag
      781 ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc
      841 gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt
      901 tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg
      961 gggggttgtc gggcagcagc acggggccgt cgccgatggg gaacaaaggc tgcgtgcggg
     1021 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc
     1081 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg
     1141 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg
     1201 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg
     1261 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
     1321 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
     1381 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
     1441 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
     1501 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1561 gtgtgaccgg cggctctagc gcctctgcta accatgttca tgccttcttc tttttcctac
     1621 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattcgcgg
     1681 ccgccaccat gtcaaggagc ctcactttca gcttcgacat cggttacgct tccattgggt
     1741 gggccgtgat tgctagtgcc tcccatgacg acgccgatcc ttctgtgtgt gggtgcggaa
     1801 cggtgctgtt tccaaaggat gactgtcagg cattcaaaag gagagagtat agaaggctgc
     1861 gcaggaacat ccgaagtcgc agagtccgga tcgagcggat cgggagactc cttgtgcaag
     1921 ctcagatcat cacacccgaa atgaaagaaa cctccggcca cccagcacct ttttaccttg
     1981 caagcgaggc cctgaaaggg catcggacac tggctccaat tgaattgtgg cacgtgctta
     2041 ggtggtatgc acacaaccga gggtatgaca acaacgcgtc ttggtctaac tctctctcag
     2101 aagacggagg taacggggaa gatactgagc gcgtcaaaca cgctcaggat ctgatggaca
     2161 agcacggaac cgcaaccatg gccgaaacca tctgtcggga gcttaagctg gaggagggaa
     2221 aggcggatgc ccccatggag gtgagtacgc cggcttacaa aaatctgaat actgcgtttc
     2281 ccaggctgat cgttgagaag gaggtgagac ggattctcga gctgagtgcg cccctgatcc
     2341 cagggctgac agccgagatc attgagctga tcgcccaaca ccatcctttg acgacagaac
     2401 agcggggggt gctgctgcag cacggtatca agctcgcacg aaggtatcgc ggcagtctcc
     2461 tttttggcca actgatacca cggttcgaca accgaattat cagcagatgc cctgtaacat
     2521 gggcgcaggt ctatgaggcc gagctgaaga aagggaatag cgagcagtcc gctagagaac
     2581 gagctgaaaa attgtctaag gtccccacgg ccaattgccc tgaattttac gagtatcgca
     2641 tggcccgcat tctgtgcaac atccgggccg acggtgaacc tctctctgcg gaaatcagaa
     2701 gggagctgat gaatcaggca cgccaagagg ggaaactgac caaggcctcc ctggagaaag
     2761 ccataagttc tagactgggg aaggaaactg aaaccaacgt cagcaattac ttcacccttc
     2821 atcccgactc cgaagaggca ctgtacttga atcccgccgt tgaggtcctg cagcgctcag
     2881 ggattgggca gatactttca cccagcgtgt acagaattgc tgccaatcgc ctgagacgag
     2941 gaaaaagtgt gacacccaat tacctgctca acctcctgaa gtccaggggt gagtcaggag
     3001 aggccctgga aaagaagatt gagaaagaga gtaagaagaa agaggcagat tatgccgaca
     3061 ctcctctgaa acctaagtac gccaccggcc gcgccccgta tgcacgcacc gttctcaaga
     3121 aggtggtcga ggaaatactg gatggggagg atcctacccg gccagccagg ggcgaggctc
     3181 accctgacgg ggaattgaaa gcccacgatg ggtgtctgta ttgtcttctt gataccgact
     3241 ccagcgtaaa tcagcaccag aaggagcggc ggctggatac catgactaat aaccatctcg
     3301 tgagacacag gatgctcatc ctcgacagac tgctcaaaga cctcattcag gactttgccg
     3361 atggacaaaa ggacaggatc tccagggtgt gcgtcgaagt cggcaaagaa ctgacaacat
     3421 tttcagccat ggactctaag aagattcaga gagagctcac gcttaggcag aagtcacaca
     3481 ctgacgctgt caatagactc aagagaaagc tgcctggaaa agcccttagc gcaaatctga
     3541 taaggaagtg ccggatcgcc atggacatga actggacctg cccctttacg ggtgcgacgt
     3601 acggagatca tgagctggag aacctggaac tggagcacat cgttcctcac agctttcgcc
     3661 aatctaacgc gctgtcatcc ctggtgctca cctggcctgg cgtcaatcgc atgaaggggc
     3721 aacggacagg atacgacttc gttgagcagg aacaggaaaa tcccgtgcca gataagccca
     3781 acctgcacat ttgttccttg aataactatc gcgaactggt ggagaaactc gacgataaga
     3841 aagggcatga ggacgacaga aggcgcaaga agaaacggaa ggcccttctt atggtgagag
     3901 gattgagcca caagcatcag tcacagaatc atgaggctat gaaggagatt ggaatgaccg
     3961 agggcatgat gacacagtcc tcacatctta tgaagctcgc ttgtaaatca atcaagacat
     4021 cactgcccga cgcacatatc gatatgatcc ccggcgccgt taccgccgag gtgagaaagg
     4081 cctgggatgt attcggagtt ttcaaggagc tctgtcccga ggccgcagat cctgatagcg
     4141 ggaagatcct caaggaaaac ctgcgctcac ttacgcatct gcatcacgca cttgatgcct
     4201 gcgtactggg gctgatccca tatattatcc ctgctcatca taatggtctt ctgcggcgag
     4261 tgctggctat gcgacgcatc ccagagaagc tgatacccca ggtgcggcct gtcgcaaatc
     4321 agcggcatta cgtgttgaat gacgatggac gaatgatgct gagggacctc agcgcctccc
     4381 tgaaggagaa catccgcgag cagctgatgg agcaacgggt gatccagcat gtgccggcag
     4441 acatgggcgg agccctgctg aaagaaacta tgcaaagagt cttgtctgtg gatggatctg
     4501 gagaagacgc aatggtaagt ctgtccaaga agaaggacgg caaaaaggag aagaaccagg
     4561 tgaaggcctc caaattggtc ggggtgttcc ccgaagggcc atcaaaactt aaggcactta
     4621 aggctgccat cgagatcgac ggcaactacg gagtggcatt ggatccgaaa cctgtcgtga
     4681 tcagacacat caaagtgttt aaaagaatca tggcgctgaa ggagcagaac ggtggtaagc
     4741 cggttaggat cctgaagaag ggaatgttga ttcacctgac ctcctcaaaa gatcctaagc
     4801 acgccggggt ctggagaatt gagtctatcc aggattcaaa ggggggggtc aagctcgacc
     4861 tgcagagggc tcattgcgcc gtgccaaaaa ataagacaca cgaatgcaat tggagggagg
     4921 tcgacctgat ctcactgctc aaaaagtacc agatgaagag gtacccaact tcatatacgg
     4981 gcaccccaag aagcagggct gaccccaaga agaagaggaa ggtgaggtcc ggcggcggag
     5041 agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc ccaatggtga
     5101 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     5161 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     5221 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     5281 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     5341 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     5401 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     5461 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     5521 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     5581 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     5641 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     5701 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     5761 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taactgcagc
     5821 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5881 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5941 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     6001 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     6061 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat
     6121 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6181 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6241 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     6301 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6361 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6421 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6481 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6541 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6601 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6661 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6721 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6781 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6841 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6901 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6961 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     7021 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     7081 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     7141 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     7201 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     7261 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     7321 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7381 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7441 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7501 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7561 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7621 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7681 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7741 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7801 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7861 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7921 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7981 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     8041 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     8101 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     8161 cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt
     8221 cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     8281 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     8341 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     8401 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     8461 cggggtcatt agttcatagc ccatatatgg agttccgcgt t
//
