LOCUS       sequence-151455-        5638 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eGFP with ecDHFR along with an U6 promoter driven gRNA 
            which cleaves the plasmid in-cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5638)
  AUTHORS   Talas A, Kulcsar PI, Weinhardt N, Borsy A, Toth E, Szebenyi K, 
            Krausz SL, Huszar K, Vida I, Sturm A, Gordos B, Hoffmann OI, 
            Bencsura P, Nyeste A, Ligeti Z, Fodor E, Welker E
  TITLE     A convenient method to pre-screen candidate guide RNAs for 
            CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 
            'self-cleaving' GFP-expression plasmid.
  JOURNAL   DNA Res. 2017 Jun 30. doi: 10.1093/dnares/dsx029.
  PUBMED    28679166
REFERENCE   2  (bases 1 to 5638)
  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: "DNA Res. 
            2017 Jun 30. doi: 10.1093/dnares/dsx029."
COMMENT     Sequence Label: pSc1-DD
FEATURES             Location/Qualifiers
     source          1..5638
                     /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      601..610
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             613..1089
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             join(1138..1140,1141..1851)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1138..1140,1141..1851)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1180..1201)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1441..1460)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1788..1809
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1852..1917
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2041..2162
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2078..2097)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2132..2151
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2169..2624)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2306..2327)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2518..2537
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2651..2755
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2757..3114
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2757..2948
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2780..2800)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2965..3100
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3149..3943
                     /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             3149..3943
                     /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(3203..3222)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3813..3832
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4131..4150)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4175..4222
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4438..4457
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4536..4611
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      4902..5490
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5391..5410
                     /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 gctagcggcc
      601 gccaccatgg ccatgatcag tctgattgcg gcgttagcgg tagattacgt tatcggcatg
      661 gaaaacgcca tgccgtggaa cctgcctgcc gatctcgcct ggtttaaacg caacacctta
      721 aataaacccg tgattatggg ccgccatacc tgggaatcaa tcggtcgtcc gttgccagga
      781 cgcaaaaata ttatcctcag cagtcaaccg agtacggacg atcgcgtaac gtgggtgaag
      841 tcggtggatg aagccatcgc ggcgtgtggt gacgtaccag aaatcatggt gattggcggc
      901 ggtcgcgtta ttgaacagtt cttgccaaaa gcgcaaaaac tgtatctgac gcatatcgac
      961 gcagaagtgg aaggcgacac ccatttcccg gattacgagc cggatgactg ggaatcggta
     1021 ttcagcgaat tccacgatgc tgatgcgcag aactctcaca gctattgctt tgagattctg
     1081 gagcggcgag gatcccttgc agaggcggct gctaaagcgg cagcgggctc gagtgtggtg
     1141 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1201 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1261 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1321 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1381 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1441 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1501 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1561 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1621 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1681 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1741 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1801 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggactc
     1861 agatctcgag ctcaagcttc gaattctgca gtcgacggta ccgcgggccc gggatccacc
     1921 ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     1981 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     2041 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2101 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     2161 taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     2221 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     2281 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     2341 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     2401 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     2461 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2521 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     2581 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     2641 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2701 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga
     2761 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2821 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2881 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2941 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3001 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     3061 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga
     3121 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     3181 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     3241 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     3301 cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc
     3361 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     3421 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     3481 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     3541 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     3601 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     3661 cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     3721 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     3781 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     3841 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     3901 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     3961 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     4021 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4081 cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg
     4141 aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg
     4201 cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     4261 gataccccac gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
     4321 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
     4381 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
     4441 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
     4501 aaaggacgaa acaccggcgc aacgcgatcg cgtaagtttt agagctagaa atagcaagtt
     4561 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttcca
     4621 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     4681 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     4741 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4801 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4861 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4921 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4981 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5041 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5101 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5161 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5221 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5281 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5341 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5401 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5461 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5521 ctggcctttt gctggccttt tgctcacatg cacaggcgca acgcgatcgc gtaagggttg
     5581 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcat
//
