LOCUS       sequence-372346-        5049 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a modified GFP (mGFP) with short half-life.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5049)
  AUTHORS   Mishra T, Bhardwaj V, Ahuja N, Gadgil P, Ramdas P, Shukla S, Chande 
            A
  TITLE     Improved loss-of-function CRISPR-Cas9 genome editing in human cells 
            concomitant with inhibition of TGF-beta signaling.
  JOURNAL   Mol Ther Nucleic Acids. 2022 Mar 8;28:202-218. doi: 
            10.1016/j.omtn.2022.03.003. eCollection 2022 Jun 14.
  PUBMED    35402072
REFERENCE   2  (bases 1 to 5049)
  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: "Mol Ther 
            Nucleic Acids."; date: "2022-03-8"; volume: "28:202-218. doi"; 
            pages: " 10.1016/j.omtn.2022.03.003. eCollection 2022 Jun 14"
COMMENT     Sequence Label: pmGFP
FEATURES             Location/Qualifiers
     source          1..5049
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      635..644
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      989..998
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(995..997,998..1000,1001..1714)
                     /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(995..997,998..1000,1001..1711)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1040..1061)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1301..1320)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1648..1669
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1837..1958
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1874..1893)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1928..1947
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1965..2420)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2102..2123)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2314..2333
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2447..2551
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2553..2910
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2553..2744
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2576..2596)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2761..2896
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2945..3739
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2945..3739
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2999..3018)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3609..3628
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3927..3946)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3971..4018
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4347..4935
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4836..4855
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attcgccacc atggcgctcc gagtcaccag
      661 gaactcgaaa attaatgctg aaaataaggc gaagatcaac atggcaggcg caaagcgcgt
      721 tcctacggcc cctgctgcaa cctccaagcc cggactgagg ccaagaacag ctcttgggga
      781 cattggtaac aaagtcagtg aacaactgca ggccaaaatg cctatgaaga aggaagcaaa
      841 accttcagct actggaaaag tcattgataa aaaactacca aaacctcttg aaaaggtacc
      901 tatgctggtg ccagtgccag tgtctgagcc agtgccagag ccagaacctg agccagaacc
      961 tgagcctgtt aaagaggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt
     1021 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1081 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1141 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1201 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1261 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1321 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1381 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1441 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1501 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1561 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     1621 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     1681 gatcactctc ggcatggacg agctgtacaa gtaaagcggc cgcgactcta gatcataatc
     1741 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     1801 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     1861 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     1921 tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa
     1981 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     2041 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     2101 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     2161 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     2221 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     2281 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     2341 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     2401 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2461 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2521 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     2581 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2641 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2701 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     2761 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     2821 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     2881 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     2941 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     3001 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     3061 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     3121 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     3181 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3241 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3301 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3361 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3421 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     3481 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     3541 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3601 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     3661 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     3721 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     3781 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     3841 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     3901 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     3961 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
     4021 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     4081 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     4141 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
     4201 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4261 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4321 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4381 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4441 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     4501 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4561 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4621 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4681 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4741 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4801 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4861 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4921 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4981 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5041 gccatgcat
//
