LOCUS       sequence-263890-        6805 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 6805)
  TITLE     ENCODE collection: CRISPR constructs
REFERENCE   2  (bases 1 to 6805)
  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: pZNF8-donor
FEATURES             Location/Qualifiers
     source          1..6805
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(19..36)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(19..35)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(28..50)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(244..263)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     363..385
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(423..441)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        509..613
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(614..682,683..1474)
                     /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     complement(832..851)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1645..2233
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2134..2153
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2387..2404
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2521..2542
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2557..2562,2563..2580,2581..2587)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2595..2611
                     /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)."
     primer_bind     2600..2622
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2619..2635
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2619..2635
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3554..3574
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3587..3631
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3671..3694
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(3722..3724,3725..3727,3728..4441)
                     /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(3767..3788)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4028..4047)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(4479..5052,5053..5055,5056..5064)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4645..4662)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4872..4891
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5146..5949
                     /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(5209..5228)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5819..5838
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
       61 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
      121 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct
      181 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
      241 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
      301 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
      361 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
      421 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
      481 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
      541 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
      601 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
      661 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
      721 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
      781 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
      841 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
      901 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
      961 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     1021 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     1081 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     1141 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     1201 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     1261 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     1321 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     1381 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     1441 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     1501 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1561 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1621 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     1681 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1741 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     1801 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1861 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1921 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1981 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2041 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2101 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2161 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2221 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2281 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2341 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     2401 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     2461 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     2521 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     2581 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     2641 ttacgccaag cttagacctt atatgtgcaa ggagtgtggg aaagccttca gccagaactc
     2701 ctccctcgtc cagcatgagc gcatccacac tggagacaag ccctacaagt gtgccgaatg
     2761 tgggaagtct ttctgccata gtacacacct taccgtccat cggaggattc acactgggga
     2821 gaagccctat gagtgtcagg actgtgggag ggccttcaac cagaactcct ccctggggcg
     2881 gcacaagagg acacacactg gggagaagcc atacacctgc agtgtgtgtg ggaaatcctt
     2941 ctctcggacc acttgccttt tcctgcacct gagaactcac accgaggaga ggccctacga
     3001 gtgtaaccac tgcgggaagg gcttcaggca cagctcatcc ctggcccagc accagcggaa
     3061 gcacgcgggg gagaagccct ttgagtgccg ccagaggctg atctttgagc agacgccagc
     3121 tctcacaaag catgaatgga cagaagccct gggctgtgac ccacctttga gtcaagatga
     3181 gaggactcac cgaagcgaca gacccttcaa atgtaatcag tgtgggaagt gtttcattca
     3241 gagctctcac ctcatccggc accagataac tcacaccaga gaggagcagc cccatgggcg
     3301 aagccggcgg cgtgaacaat cctcgagcag gaactcacac ctggttcagc atcaacaccc
     3361 gaactccaga aagagctctg caggcggagc aaaggcaggg cagccggaaa gcagagccct
     3421 ggctttgttt gacatccaaa aaatcatgca agagaaaaac cctgtgcacg ttattggggt
     3481 ggaagagcct tctgtgggtg cttccatgtt atttgacatc agagaatcca cagggattcc
     3541 aactactgca agcgagaatc tttattttca gggcgccgcc aaattcaaag aaaccgctgc
     3601 tgctaaattc gaacgccagc acatggacag cggaggtgga ggttcgagcg gtccctcggg
     3661 ttcgtcgagc ctggaagttc tgttccaggg gcccctgtcg tcgagcggtc cctcgggttc
     3721 gatggtgagc aaaggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     3781 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     3841 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     3901 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     3961 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     4021 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     4081 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     4141 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     4201 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     4261 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     4321 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     4381 ccttaaggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     4441 atgatctaga gtcgagttaa ttaagaattc cgcccccccc cctctccctc ccccccccta
     4501 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     4561 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     4621 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcagggtctg ttgaatgtcg
     4681 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     4741 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     4801 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     4861 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     4921 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     4981 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     5041 cacgatgata atatggccac aacctgttac attgcacaag ataaaaatat atcatcacga
     5101 acagtaaaac tgtctgctta cataaacagt aatacaaggg gtgttatggg atcggccatt
     5161 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     5221 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     5281 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     5341 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     5401 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     5461 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     5521 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     5581 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5641 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     5701 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5761 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5821 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5881 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5941 ttcttctgag agagaaactt tgctgatgac ttttaaccac aagtaaaaaa tgtggtaagt
     6001 ccacatagtg tactcatgga aggaggggct gggggtagaa atgtcatggg tgacttctga
     6061 ctttctaagg aaatgatgct tcccaagcac ccgaggttgg ttggtcccaa atctatcaaa
     6121 ctcagtgccc tctttagcga catattttgt gacattcctt ccattacacc acagtgagtt
     6181 cacaggtaat ataacctacc cacctgtgta atgtcaaaaa aaatcaatat gcggccccat
     6241 tttgtaaagg atcattaaaa tgaaagtaat ttatggtaga gtaaattgta gagtaacgtg
     6301 tactgacttg gtgcagtatt tctccttgtc actgtgggcc caattttgcc aggagaccta
     6361 gggcaatagc ctgttgcttg tggactggta gagggtatga tgttgaccat ggcaatactg
     6421 tgaggggcct tcccatgtgg aacgtgacct tccaaagtag tgactaactc ttggaaaagt
     6481 ttttaacaac ataatatgct attgcatttc tgggaaattc tgggtaaatt ccaagcatgc
     6541 agcaattagt ttgtgcttat gtgtgaagtc cagttaggtt ctcagttcat accctgaaaa
     6601 ctgcttattc cacatctgtt gatgtggtcc actgggggca gttctgttcc tcaccatctt
     6661 ggcagcatgc atggctcctg cccacatttc aaccacaccc ccaacccaat tacgtgacca
     6721 gcccccacga aatttcacag cattccgtgc aggggagtgt cacagctcct cttgaaaaac
     6781 atctggtgtt gctatgagcc agaag
//
