LOCUS       sequence-347402-        7273 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 7273)
  TITLE     ENCODE collection: CRISPR constructs
REFERENCE   2  (bases 1 to 7273)
  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: pZNF608-donor
FEATURES             Location/Qualifiers
     source          1..7273
                     /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             3868..3888
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3901..3945
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3985..4008
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(4036..4038,4039..4041,4042..4755)
                     /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(4081..4102)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4342..4361)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(4793..5366,5367..5369,5370..5378)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4959..4976)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5186..5205
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5460..6263
                     /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(5523..5542)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6133..6152
                     /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 tcggtacccg gaggaagcaa gtacaggatt tttagaccaa
     2701 aaacttaact ccttgggttt tatatcattg taatgaatgt gtttattatg ccaatataag
     2761 ttgcttcaca tgcaacttaa attccatgaa cctcattctg taagggttac atagattagt
     2821 aattaactga ttggtttttc agacagttga aattttcttg ggttatcctt acggcatttc
     2881 taaaataatt caccatgact ccctttgttt tattatgttt aaagataaag tcgcaaatgg
     2941 agacatttat tttcataatg ctaaaggtaa tgtttgttct ttgatatttt aaattattcc
     3001 ctaagtttta tgaaggaaaa tgatttaaag gaattaaatt attaatatta taaaattaat
     3061 atttttcaca atgtatatca ttactttatg tttattcact ttctccggaa ttgaagtcca
     3121 catttgaatg aaattaaaat aagactatcg gtcttatggt tttctggttt acagtgccaa
     3181 atgttgatag aggctgggca tgatggccca tgcctgtagt cccaccactt tgggaggctc
     3241 atgcaggtgg atcacctgag gtcgggagtt ccagactagc ctggccaaca tggtgaaacc
     3301 ccatctctaa taaaaataca aaaattagct gggcgtggtg gtgtgcgtct gtaattccag
     3361 ctactaggga ggctgaggca tgagaattgc tggaacccag gaggtggagg ttgcagtaag
     3421 ctgagattgc accattgcat tccagcctag gtgatggagt gagactctgt ctcaaaacta
     3481 aaaaaaaaaa aaaactgttg gccgggtgcg gtggctcatg cctataatcc cagcactttg
     3541 ggaggccgag gtgggcagat cacctgaggt caggagcttg agaccagtct gaccaacatg
     3601 gagaaacccc ctctctacta aaaacataaa attagtcagg catggtggca catgcctata
     3661 atcccagcta ctcgggaggc tgaggcagga gaatcacttg aacccaggag gcagaggttg
     3721 cggtgagccg agattgagcc gtggcactct agcctgggca acaagagcaa aactccgact
     3781 caaaaaacaa acaaacaaaa tgttgataga accgtgtgct aaggtttgct tttcttttgc
     3841 agagaaggga ttccaactac tgcaagcgag aatctttatt ttcagggcgc cgccaaattc
     3901 aaagaaaccg ctgctgctaa attcgaacgc cagcacatgg acagcggagg tggaggttcg
     3961 agcggtccct cgggttcgtc gagcctggaa gttctgttcc aggggcccct gtcgtcgagc
     4021 ggtccctcgg gttcgatggt gagcaaaggc gaggagctgt tcaccggggt ggtgcccatc
     4081 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     4141 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     4201 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     4261 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     4321 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     4381 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     4441 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     4501 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     4561 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4621 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     4681 cgcgatcaca tggtccttaa ggagttcgtg accgccgccg ggatcactct cggcatggac
     4741 gagctgtaca agtaatgatc tagagtcgag ttaattaaga attccgcccc cccccctctc
     4801 cctccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt
     4861 ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg
     4921 ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaggg
     4981 tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc
     5041 tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca
     5101 aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag
     5161 ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa
     5221 ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt
     5281 tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt
     5341 tttcctttga aaaacacgat gataatatgg ccacaacctg ttacattgca caagataaaa
     5401 atatatcatc acgaacagta aaactgtctg cttacataaa cagtaataca aggggtgtta
     5461 tgggatcggc cattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     5521 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     5581 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     5641 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     5701 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     5761 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     5821 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     5881 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     5941 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     6001 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     6061 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     6121 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     6181 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     6241 gccttcttga cgagttcttc tgagtccaag attggacatg tacattgtca tgtgactgga
     6301 tcacatgggg aagcgcttta tacagacatc agttccatgg tgttaatttg aaatgcctta
     6361 atggcaggca aaaaccagtc atttcatttt tgtaaattac agattttatc acagagtaac
     6421 aaatgttgct gtaattaaac ttctgtttta tatatatata cgtatacata tctgtatata
     6481 tacatatata tatatataaa tataaatata ttctttactt tttgtatcag taaccaggct
     6541 cgcacacaca gggtctgctg ccacgtcgca aaccactcag taaaggaaat taaaaatgta
     6601 tttgtcatgt taaaaagtgt tagccacaaa aggctactta ctttcttttc cttttccttt
     6661 aaaattgtaa ataaataatg ttatgtatca gtgtgcctga accttgcata tccttcacat
     6721 atttcccata agcccctcag aaaggctaac tgtgatgatg acactttggt acaatttaga
     6781 tgtctatttg gtggctcctg ttatagacgc accagtgtaa aatgttcctg tagtcactgt
     6841 ttgtacttgt gtattgtgga aaaagtactt ttggaaggca tggggttgcc agtaccacaa
     6901 aaactgtgtt gtatataatg taaagattaa aatggggaaa taatgagcat ctgtgaataa
     6961 tgaggtgtca ttttttgata atcttgaatg gcaaataacc caatagcctg cgtaccagag
     7021 acatctgtga ttgttctatt acttgaatag ttctacagtc tttttttttt tttttaatgt
     7081 ttacaattat ccagttttag aagagagaga ctttaacgcc attgcctggt tacttgtttt
     7141 atgctgtaat ctagtttatt ttatgtaaaa tgtatattga atgctttcct tttttatacc
     7201 tgccttaaat aatttggcaa tcagaattaa aaagtttttt ttttttttaa ataatggctt
     7261 cttgtctgtc tca
//
