LOCUS       sequence-274870-        7394 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 7394)
  TITLE     ENCODE collection: CRISPR constructs
REFERENCE   2  (bases 1 to 7394)
  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: pTSHZ1-donor
FEATURES             Location/Qualifiers
     source          1..7394
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(265..281)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(265..281)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(278..300)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    289..305
                     /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(313..319,320..337,338..343))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    358..379
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(496..513)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(667..1255)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(747..766)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1426..2217,2218..2286))
                     /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     2049..2068
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2287..2391)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2459..2477
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2515..2537)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2637..2656
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2850..2872
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2864..2881
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2865..2881
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4115..4135
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4148..4192
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             4232..4255
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(4283..4285,4286..4288,4289..5002)
                     /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(4328..4349)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4589..4608)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(5040..5613,5614..5616,5617..5625)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5206..5223)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5433..5452
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5707..6510
                     /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(5770..5789)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6380..6399
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 cctttccttg taaattgcca gcatcagctt aaaaactcct gtatgttaca tatcgtacca
       61 ttttaatctc ttcaactttc tttgtacctt ctggctgtat gctttctctt ttaacttttt
      121 atattgtcat tttatattaa atttctgtgt atataatgta aaaccacaat ttttgaataa
      181 aatttagttc ctgcagcttg atttaaatag aggactttta ctggcacctg cgacctgcag
      241 gcatgcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
      301 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
      361 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
      421 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
      481 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
      541 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      601 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
      661 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      721 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
      781 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
      841 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
      901 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
      961 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     1021 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     1081 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     1141 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     1201 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     1261 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     1321 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     1381 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     1441 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     1501 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     1561 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     1621 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     1681 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     1741 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     1801 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     1861 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     1921 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     1981 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     2041 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     2101 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     2161 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     2221 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     2281 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     2341 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     2401 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     2461 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     2521 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     2581 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     2641 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     2701 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
     2761 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     2821 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     2881 tgaattcgag ctcggtaccc tcccgaaaac ggaggaagtc agcggcaaac cacagaagaa
     2941 gggccctgag gccgagactg ggaaggccaa aaaggaggga ccgctggacg ttcacacccc
     3001 aaatggcaca gagcctctca aagcaaaggt caccaacggc tgtaacaacc tggggatcat
     3061 catggaccac tcaccggagc cttccttcat caacccgctg agcgctttgc agtccatcat
     3121 gaacacccac ctgggcaagg tgtccaagcc cgtgagtccc tcgctggacc cgctggcgat
     3181 gctgtacaag atcggcaaca gcatgctgga caagccggtg taccccgcca cccctgtgaa
     3241 gcaggccgat gccatcgacc gctactatta tgaaaacagc gaccagccca ttgacttaac
     3301 caagtccaag aacaagccgc tggtgtccag cgtggctgat tcggtggcat cacctctgcg
     3361 ggagagcgca ctcatggaca tctccgacat ggtgaaaaac ctcacaggcc gcctgacgcc
     3421 caagtcctcc acgccctcca cagtttcaga gaagtccgat gctgatggca gcagctttga
     3481 ggaggcgttg gacgagctgt caccggtcca caagaggaag ggccggcagt ccaactggaa
     3541 cccgcagcac cttctcatcc tgcaggccca gttcgcctcg agcttgcggg agaccacaga
     3601 gggcaagtac atcatgtcgg acttgggccc gcaggagagg gtgcacatct cgaagtttac
     3661 tgggctctcc atgaccacca tcagccactg gctggccaat gtgaagtacc agttgaggag
     3721 gacaggggga acgaaattcc taaagaacct ggacacaggg catcctgttt tcttttgcaa
     3781 cgattgtgcc tctcagttca gaactgcttc tacatacata agtcatttgg agacacactt
     3841 gggcttcagc ctgaaggatc tctccaagct gccactcaat cagattcaag aacagcagaa
     3901 tgtttcgaaa gtcctcacca acaaaactct gggcccactg ggggccaccg aggaagactt
     3961 gggctccaca ttccaatgta agctctgcaa ccggactttt gcgagcaagc acgcagtcaa
     4021 actgcacctt agtaagaccc acggcaagtc tcccgaggac cacctgatct atgtgactga
     4081 gttggagaaa caggggattc caactactgc aagcgagaat ctttattttc agggcgccgc
     4141 caaattcaaa gaaaccgctg ctgctaaatt cgaacgccag cacatggaca gcggaggtgg
     4201 aggttcgagc ggtccctcgg gttcgtcgag cctggaagtt ctgttccagg ggcccctgtc
     4261 gtcgagcggt ccctcgggtt cgatggtgag caaaggcgag gagctgttca ccggggtggt
     4321 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     4381 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     4441 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     4501 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     4561 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     4621 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     4681 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     4741 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     4801 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     4861 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     4921 cgagaagcgc gatcacatgg tccttaagga gttcgtgacc gccgccggga tcactctcgg
     4981 catggacgag ctgtacaagt aatgatctag agtcgagtta attaagaatt ccgccccccc
     5041 ccctctccct ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     5101 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     5161 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     5221 tgcagggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa
     5281 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     5341 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     5401 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     5461 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca
     5521 catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg
     5581 acgtggtttt cctttgaaaa acacgatgat aatatggcca caacctgtta cattgcacaa
     5641 gataaaaata tatcatcacg aacagtaaaa ctgtctgctt acataaacag taatacaagg
     5701 ggtgttatgg gatcggccat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     5761 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     5821 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     5881 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     5941 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     6001 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     6061 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     6121 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     6181 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     6241 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     6301 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     6361 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     6421 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     6481 ttctatcgcc ttcttgacga gttcttctga gtccgtccag gtatgcaaga gaccgcggaa
     6541 cattgcacta aacgtcgtcg agctgcacta ggcctggcct gagcctctga aatcagtctt
     6601 tcctttgttg ctggcccgcc tctctggacc ttggttttct tacacatatt ttgtatattt
     6661 atatgctctc tgtccgatct gtgcatgtta tttttctttt tccgtgagtc aaagtctgac
     6721 ctttattttc aacatctgtt cttggtgtta agctatcttt tgtaggaaat agtggggcac
     6781 actactcaga gacattattt agcagtaaag aaagacacaa ataacaatga taaaaagaca
     6841 tcctaaaatg gtgaagttgc catgacaata aaggtcatag aacctggtag tgtcaaattt
     6901 aaccctttga ggactgtaat tgcatttctg tgcctttcac ttgaaaaaaa aaaaagaaga
     6961 aaaaaaggct taaaggcatt tcattcagat caaaagctgt gtcagacaca aaacttattt
     7021 aataattaaa aaatgagctt ttatatgcag aactggtttg tgaggaaaca tgcatgcagc
     7081 tgtgggaaga tagaaagctg tctaggactc acggggttcg gaagccacac ctctacatgt
     7141 ctaaaacaaa aaacaacaac aacagatacc ccactcgcca ctatgcccaa accctctgga
     7201 tgctgaaaaa tgccactttc ggcaaaaaaa aaagtatgca agctattatt taaaaatgtg
     7261 taaaaatgct atttcttttt tcctgtaaaa tattgcagtt tatagttatt tacaaatgta
     7321 agctttggta caagctgacc tttctatagc gtgctgcatt ggtaaatgaa aaaaaatggg
     7381 gcaaacgttg gagt
//
