LOCUS       sequence-274812-        7134 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR donor plasmid to create GFP fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7134)
  TITLE     ENCODE collection: CRISPR constructs
REFERENCE   2  (bases 1 to 7134)
  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"
COMMENT     Sequence Label: pTOX-donor
FEATURES             Location/Qualifiers
     source          1..7134
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(34..50)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(34..50)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(47..69)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    58..74
                     /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(82..88,89..106,107..112))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    127..148
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(265..282)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(436..1024)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(516..535)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1195..1986,1987..2055))
                     /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     1818..1837
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2056..2160)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2228..2246
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2284..2306)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2406..2425
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2619..2641
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2633..2650
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2634..2650
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3722..3742
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3755..3799
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3839..3862
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(3890..3892,3893..3895,3896..4609)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3935..3956)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4196..4215)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(4647..5220,5221..5223,5224..5232)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4813..4830)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5040..5059
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5314..6117
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5377..5396)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5987..6006
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gacctgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
       61 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
      121 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
      181 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
      241 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
      301 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      361 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      421 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      481 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      541 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      601 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
      661 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      721 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      781 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      841 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
      901 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      961 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1021 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1081 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     1141 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     1201 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     1261 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     1321 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     1381 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     1441 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     1501 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     1561 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     1621 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     1681 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     1741 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     1801 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     1861 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     1921 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     1981 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     2041 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     2101 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     2161 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     2221 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     2281 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     2341 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     2401 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     2461 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     2521 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     2581 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     2641 gacggccagt gaattcgagc tcggtaccca agaagcagat ggtccctttg tgactgaatg
     2701 aagaaaaaga ggtttgagaa caatatgtga ataggtgtag tctaataaaa cactcagcca
     2761 cctgctgcct gaatccttca aaaaagattc tgttgcatat tctggggatt aactattaat
     2821 gtatccttag gtttgataga aaacataaac tttgaggggt ttaggccctc ttctcccttt
     2881 gttttgattg agttggctcc tctatatttg aaattgtttc tcaattcaat ttatctgctc
     2941 aagggtataa ttaaaggttt tctgactcat ggctaaatga aggaaaggta agaagaatgg
     3001 acttgtgggc atttcttttc tacctttgtt ttaaattttc tgggctcttc cgacaggctt
     3061 gcccttgcag agacagaagc aagaggccta gtgtgtctgc aaagaatgaa taaatattta
     3121 ggtgcttttc ttcttaaaat gcattttgat ccttatttgc ggacttgctt tccctttcag
     3181 ggatttactc ttcaacccga ctatcagact attatcaatc ctacatctac agctgcacaa
     3241 gttgtcaccc aggcaatgga gtatgtgcgt tcggggtgca gaaatcctcc cccacaaccg
     3301 gtggactgga ataacgacta ctgcagtagt gggtaagcgg ccggtcatcg ccacctggtg
     3361 gtggacagcg ctcatagcat gcatatcgtc cctagttgat cgttttcttg tttccaaaaa
     3421 catgatccgg gatagtttca cgttcatgaa ggcagaagac accagaaatt gttaagcagt
     3481 cttctagctc ttccaagaat aaatgccttt agctcacttc tcacttcaga tgtaggttaa
     3541 ctaattgcag tttcccagtg aacagctcaa gctggagcaa ataattcaaa gacatccatc
     3601 cccccattgt cacctgtagc acaggttgtc ctgtaacaag gaactgtctt ttttctcttc
     3661 ctacaggggc atgcagaggg acaaagcact gtaccttact gggattccaa ctactgcaag
     3721 cgagaatctt tattttcagg gcgccgccaa attcaaagaa accgctgctg ctaaattcga
     3781 acgccagcac atggacagcg gaggtggagg ttcgagcggt ccctcgggtt cgtcgagcct
     3841 ggaagttctg ttccaggggc ccctgtcgtc gagcggtccc tcgggttcga tggtgagcaa
     3901 aggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     3961 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     4021 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     4081 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     4141 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     4201 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     4261 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     4321 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     4381 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     4441 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     4501 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc ttaaggagtt
     4561 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaat gatctagagt
     4621 cgagttaatt aagaattccg cccccccccc tctccctccc cccccctaac gttactggcc
     4681 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     4741 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     4801 ggggtctttc ccctctcgcc aaaggaatgc agggtctgtt gaatgtcgtg aaggaagcag
     4861 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     4921 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     4981 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     5041 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     5101 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa
     5161 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat
     5221 atggccacaa cctgttacat tgcacaagat aaaaatatat catcacgaac agtaaaactg
     5281 tctgcttaca taaacagtaa tacaaggggt gttatgggat cggccattga acaagatgga
     5341 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     5401 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     5461 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     5521 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     5581 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     5641 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     5701 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     5761 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     5821 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     5881 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     5941 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     6001 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     6061 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagtc
     6121 gaatctgaac acctcttctt tccactgagg aattcaggga agtgttttca ccatggattg
     6181 ctttgtacag tcaaggcagt tctccatttt attagaaaat acaagttgct aagcacttag
     6241 gaccatttga gcttgtgggt cacccactct ggaagaaata gtcatgcttc tttattattt
     6301 ttttaatcct ttatggacat tgtttttctt cttccctgaa ggaaatttgg accattcaga
     6361 ttttatgtcg gttttttgct gtgaagtgct gcgctctagt aactgcctta gcaactgtag
     6421 atgtctcgga taaaagtcct ggattttcca ttggttttca taatgggtgt ttatatgaaa
     6481 ctactaaaga ctttttaaat ggcttgatgt agcagtcata gcaagtttgt aaatagcatc
     6541 tatgttacac tctcctagag tataaaatgt gaatgttttt gtagctaaat tgtaattgaa
     6601 actggctcat tccagtttat tgatttcaca ataggggtta aattggcaaa cattcatatt
     6661 tttacttcat ttttaaaaca actgactgat agttctatat tttcaaaata tttgaaaata
     6721 aaaagtattc ccaagtgatt ttaatttaaa aacaaattgg ctttgtctca ttgatcagac
     6781 aaaaagaaac tagtattaag ggaagcgcaa acacatttat tttgtactgc agaaaaattg
     6841 cttttttgta tcactttttg tgtaatggtt agtaaatgtc atttaagtcc ttttatgtat
     6901 aaaactgcca aatgcttacc tggtatttta ttagatgcag aaacagattg gaaacagcta
     6961 aattacaact tttacatatg gctctgtctt attgtttctt catactgtgt ctgtatttaa
     7021 tcttttttta tggaacctgt tgcgcctatt tatgaaataa taaatatagg tgtttgtaag
     7081 taaatttgtt agtatttgaa agaggtttct ttgatgtttt aacttttgct ggca
//
