LOCUS       sequence-306373-        6581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6581)
  AUTHORS   Hyodo T, Rahman ML, Karnan S, Ito T, Toyoda A, Ota A, Wahiduzzaman 
            M, Tsuzuki S, Okada Y, Hosokawa Y, Konishi H
  TITLE     Tandem Paired Nicking Promotes Precise Genome Editing with Scarce 
            Interference by p53.
  JOURNAL   Cell Rep. 2020 Jan 28;30(4):1195-1207.e7. doi: 
            10.1016/j.celrep.2019.12.064.
  PUBMED    31995758
REFERENCE   2  (bases 1 to 6581)
  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"; doi: 
            "10.1016/j.celrep.2019.12"; journalName: "Cell Rep"; date: 
            "2020-01-28- 28"; volume: "30"; issue: "4"; pages: "1195-1207.e7"
COMMENT     Sequence Label: pEGFP-IREShygro2
FEATURES             Location/Qualifiers
     source          1..6581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      950..959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(956..958,959..961,962..1672)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(956..958,959..961,962..1672)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1001..1022)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1262..1281)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1609..1630
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1673..1738
                     /label=MCS
                     /note="multiple cloning site"
     intron          1815..2044
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2027..2046
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2127..2701,2702..2704,2705..2713)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2294..2311)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2521..2540
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2739..3758
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             2739..3758
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     complement(4068..4085)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4074..4298
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4345..4364
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4424..4440)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4424..4440)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4437..4459)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4448..4464
                     /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(4472..4478,4479..4496,4497..4502))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4517..4538
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4655..4672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4826..5414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4906..4925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5585..6376,6377..6445))
                     /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     6208..6227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6446..6550)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ctgtacaggc cttaagccgg cgcgctagcg ctaccggtcg ccaccatggt
      961 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1021 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1081 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1141 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1201 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1261 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1321 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1381 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1441 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1501 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1561 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1621 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact
     1681 cagatctcga gctcaagctt cgaattctgc agtcgacggt accgcgggcc cgggatccga
     1741 tatcgtacgt taacatagat aactgatcca gtgtgctgga attaattcgc tgtctgcgag
     1801 ggccagctgt tggggtgagt actccctctc aaaagcgggc atgacttctg cgctaagatt
     1861 gtcagtttcc aaaaacgagg aggatttgat attcacctgg cccgcggtga tgcctttgag
     1921 ggtggccgcg tccatctggt cagaaaagac aatctttttg ttgtcaagct tgaggtgtgg
     1981 caggcttgag atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc
     2041 acaggtgtcc actcccaggt ccaactgcag gtcgagcatg catctagggc ggccgcacta
     2101 gaggaattcc gcccctctcc cccccccccc tctccctccc ccccccctaa cgttactggc
     2161 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     2221 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     2281 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     2341 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     2401 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     2461 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     2521 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     2581 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
     2641 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     2701 gcttgccaca acccgtacca aagatggata gatccggaaa gcctgaactc accgcgacgt
     2761 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     2821 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     2881 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     2941 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     3001 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     3061 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     3121 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     3181 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     3241 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     3301 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     3361 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     3421 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     3481 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     3541 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     3601 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     3661 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     3721 accgacgccc cagcactcgt ccgagggcaa aggaatagag tagatgccga ccgaacaaga
     3781 gctgatttcg agaacgcctc agccagcaac tcgcgcgagc ctagcaaggc aaatgcgaga
     3841 gaacggcctt acgcttggtg gcacagttct cgtccacagt tcgctaagct cgctcggctg
     3901 ggtcgcggga gggccggtcg cagtgattca ggcccttctg gattgtgttg gtccccaggg
     3961 cacgattgtc atgcccacgc actcgggtga tctgactgat cccgcagatt ggagatcgcc
     4021 gcccgtgcct gccgattggg tgcagatctc tagagctcgc tgatcagcct cgactgtgcc
     4081 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4141 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4201 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4261 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     4321 ctggggctcg agtgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4381 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4441 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4501 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4561 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4621 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4681 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4741 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4801 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4861 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4921 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4981 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5041 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5101 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacgcgact
     5161 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5221 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5281 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5341 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5401 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5461 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5521 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5581 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5641 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5701 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5761 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5821 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5881 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5941 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6001 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6061 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6121 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6181 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6241 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6301 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6361 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6421 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6481 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6541 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
