LOCUS       sequence-347890-        7374 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 7374)
  TITLE     ENCODE collection: CRISPR constructs
REFERENCE   2  (bases 1 to 7374)
  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: pID3-donor
FEATURES             Location/Qualifiers
     source          1..7374
                     /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             3787..3807
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3820..3864
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3904..3927
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(3955..3957,3958..3960,3961..4674)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLVDHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4000..4021)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4261..4280)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(4712..5285,5286..5288,5289..5297)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4878..4895)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5105..5124
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5379..6182
                     /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(5442..5461)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6052..6071
                     /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 tcggtaccct cagcttcacc gcaattaatt ttttttccct
     2701 ctggtcacaa gataattcct gacgccagtg agtctggagg tcagacgaac agcaaattgg
     2761 ggaacaaggc ggcactaatt ccttacaagt ttcccttgaa aaatctttcg cttaaaaaaa
     2821 acggggggtg gggggagctt ctttgctgtt cagggattta tgacctcgga ggagctgtgg
     2881 ctcgaaccag tgttgggcta aaggcggact ggcagggggc agggaagctc aaagatctgg
     2941 ggtgctgcca ggaaaaagca aattctggaa gttaatggtt ttgagtgatt tttaaatcct
     3001 tgctggcgga gaggcccgcc tctccccggt atcagcgctt cctcattctt tgaatccgcg
     3061 gctccgcggt cttcggcgtc agaccagccg gaggaagcct gtttgcaatt taagcgggct
     3121 gtgaacgccc agggccggcg ggggcagggc cgaggcgggc cattttgaat aaagaggcgt
     3181 gccttccagg caggctctat aagtgaccgc cgcggcgagc gtgcgcgcgt tgcaggtcac
     3241 tgtagcggga cttcttttgg ttttctttct ctttggggca cctctggact cactccccag
     3301 catgaaggcg ctgagcccgg tgcgcggctg ctacgaggcg gtgtgctgcc tgtcggaacg
     3361 cagtctggcc atcgcccggg gccgagggaa gggcccggca gctgaggagc cgctgagctt
     3421 gctggacgac atgaaccact gctactcccg cctgcgggaa ctggtacccg gagtcccgag
     3481 aggcactcag cttagccagg tggaaatcct acagcgcgtc atcgactaca ttctcgacct
     3541 gcaggtagtc ctggccgagc cagcccctgg accccctgat ggcccccacc ttcccatcca
     3601 ggtaagcctc gaagtcggga cagggctgaa cacccaggca aggatgctgc gggaccctcg
     3661 gagctcccga ttgcctcgcg taactcttcc ctcttttcct ctaatcagac agccgagctc
     3721 gctccggaac ttgtcatctc caacgacaaa aggagctttt gccacgggat tccaactact
     3781 gcaagcgaga atctttattt tcagggcgcc gccaaattca aagaaaccgc tgctgctaaa
     3841 ttcgaacgcc agcacatgga cagcggaggt ggaggttcga gcggtccctc gggttcgtcg
     3901 agcctggaag ttctgttcca ggggcccctg tcgtcgagcg gtccctcggg ttcgatggtg
     3961 agcaaaggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     4021 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     4081 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     4141 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     4201 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     4261 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     4321 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     4381 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     4441 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgtcgaccac
     4501 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     4561 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtccttaag
     4621 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaatgatct
     4681 agagtcgagt taattaagaa ttccgccccc ccccctctcc ctcccccccc ctaacgttac
     4741 tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat
     4801 attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat
     4861 tcctaggggt ctttcccctc tcgccaaagg aatgcagggt ctgttgaatg tcgtgaagga
     4921 agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca
     4981 gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac
     5041 acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt
     5101 caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca
     5161 ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt
     5221 aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg
     5281 ataatatggc cacaacctgt tacattgcac aagataaaaa tatatcatca cgaacagtaa
     5341 aactgtctgc ttacataaac agtaatacaa ggggtgttat gggatcggcc attgaacaag
     5401 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     5461 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     5521 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     5581 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     5641 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     5701 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5761 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5821 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     5881 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     5941 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     6001 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     6061 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     6121 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     6181 gagtcctcgg ccgtgtcctg acacctccag gtgagtatct cctctcttgg agagggaggt
     6241 ttaaacggca agtcctggag ttggcagacg ttttgaaaaa ttgccactca ctcggtttag
     6301 ggaaactgag gccagagagg gacaagtgac ttgcccatgg ttgcatcaaa tgaatggcag
     6361 agtcagtttc catgtgatgt gcatttaagc cttaaatgcg cctgggccct gccctcccgc
     6421 agtggccgag ggtctggcaa agtagacatg ggtccgacta aatacaagtc tttctgttcc
     6481 atgttgtata gggagctgtc ttcggcagcc ccctcccagc tagtgtcaat tccaagtagg
     6541 aggggtagcg caagctccgc ctgtggtctt tggcgccaac tgggtggggg cagcgtgggg
     6601 cgcggagtta tcagctggag gtacagacca agtttcctcc ctggcgccgg ccagtctgcg
     6661 gacggccccc gcctcggcac gctcggcgga aactgactgc tccttggtct tctttcctcc
     6721 cccgcccaga acgcaggtgc tggcgcccgt tctgcctggg accccgggaa cctctcctgc
     6781 cggaagccgg acggcaggga tgggccccaa cttcgccctg cccacttgac ttcaccaaat
     6841 cccttcctgg agactaaacc tggtgctcag gagcgaagga ctgtgaactt gtggcctgaa
     6901 gagccagagc tagctctggc caccagctgg gcgacgtcac cctgctccca ccccaccccc
     6961 aagttctaag gtcttttcag agcgtggagg tgtggaagga gtggctgctc tccaaactat
     7021 gccaaggcgg cggcagagct ggtcttctgg tctccttgga gaaaggttct gttgccctga
     7081 tttatgaact ctataataga gtatataggt tttgtacctt ttttacagga aggtgacttt
     7141 ctgtaacaat gcgatgtata ttaaactttt tataaaagtt aacattttgc ataataaacg
     7201 atttttaaac acttgtgtat atgatgacac ccgtctccat taagtactaa tgatgctttc
     7261 tcgcacatgg ccgaattttg ggagctttgg gaaagtgaac ttgcttattc tacgagaggg
     7321 aaatgaaaaa ctgcctggtt gagaggggat ggggtggaga gagaagggtt catg
//
