LOCUS       sequence-274397-        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"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1034..1054
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1067..1111
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1151..1174
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1202..1204,1205..1207,1208..1921)
                     /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(1247..1268)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1508..1527)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(1959..2532,2533..2535,2536..2544)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2125..2142)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2352..2371
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2626..3429
                     /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(2689..2708)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3299..3318
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4304..4321)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4304..4320)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4313..4335)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4529..4548)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4648..4670
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4708..4726)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4794..4898
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4899..4967,4968..5759)
                     /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(5117..5136)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5930..6518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6419..6438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6672..6689
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cttagacctt atatgtgcaa ggagtgtggg aaagccttca gccagaactc
      181 ctccctcgtc cagcatgagc gcatccacac tggagacaag ccctacaagt gtgccgaatg
      241 tgggaagtct ttctgccata gtacacacct taccgtccat cggaggattc acactgggga
      301 gaagccctat gagtgtcagg actgtgggag ggccttcaac cagaactcct ccctggggcg
      361 gcacaagagg acacacactg gggagaagcc atacacctgc agtgtgtgtg ggaaatcctt
      421 ctctcggacc acttgccttt tcctgcacct gagaactcac accgaggaga ggccctacga
      481 gtgtaaccac tgcgggaagg gcttcaggca cagctcatcc ctggcccagc accagcggaa
      541 gcacgcgggg gagaagccct ttgagtgccg ccagaggctg atctttgagc agacgccagc
      601 tctcacaaag catgaatgga cagaagccct gggctgtgac ccacctttga gtcaagatga
      661 gaggactcac cgaagcgaca gacccttcaa atgtaatcag tgtgggaagt gtttcattca
      721 gagctctcac ctcatccggc accagataac tcacaccaga gaggagcagc cccatgggcg
      781 aagccggcgg cgtgaacaat cctcgagcag gaactcacac ctggttcagc atcaacaccc
      841 gaactccaga aagagctctg caggcggagc aaaggcaggg cagccggaaa gcagagccct
      901 ggctttgttt gacatccaaa aaatcatgca agagaaaaac cctgtgcacg ttattggggt
      961 ggaagagcct tctgtgggtg cttccatgtt atttgacatc agagaatcca cagggattcc
     1021 aactactgca agcgagaatc tttattttca gggcgccgcc aaattcaaag aaaccgctgc
     1081 tgctaaattc gaacgccagc acatggacag cggaggtgga ggttcgagcg gtccctcggg
     1141 ttcgtcgagc ctggaagttc tgttccaggg gcccctgtcg tcgagcggtc cctcgggttc
     1201 gatggtgagc aaaggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1261 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1321 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1381 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     1441 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1501 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1561 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1621 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1681 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1741 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1801 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     1861 ccttaaggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     1921 atgatctaga gtcgagttaa ttaagaattc cgcccccccc cctctccctc ccccccccta
     1981 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     2041 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     2101 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcagggtctg ttgaatgtcg
     2161 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     2221 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     2281 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     2341 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     2401 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     2461 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     2521 cacgatgata atatggccac aacctgttac attgcacaag ataaaaatat atcatcacga
     2581 acagtaaaac tgtctgctta cataaacagt aatacaaggg gtgttatggg atcggccatt
     2641 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     2701 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     2761 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     2821 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     2881 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     2941 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3001 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3061 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3121 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     3181 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3241 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     3301 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     3361 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     3421 ttcttctgag agagaaactt tgctgatgac ttttaaccac aagtaaaaaa tgtggtaagt
     3481 ccacatagtg tactcatgga aggaggggct gggggtagaa atgtcatggg tgacttctga
     3541 ctttctaagg aaatgatgct tcccaagcac ccgaggttgg ttggtcccaa atctatcaaa
     3601 ctcagtgccc tctttagcga catattttgt gacattcctt ccattacacc acagtgagtt
     3661 cacaggtaat ataacctacc cacctgtgta atgtcaaaaa aaatcaatat gcggccccat
     3721 tttgtaaagg atcattaaaa tgaaagtaat ttatggtaga gtaaattgta gagtaacgtg
     3781 tactgacttg gtgcagtatt tctccttgtc actgtgggcc caattttgcc aggagaccta
     3841 gggcaatagc ctgttgcttg tggactggta gagggtatga tgttgaccat ggcaatactg
     3901 tgaggggcct tcccatgtgg aacgtgacct tccaaagtag tgactaactc ttggaaaagt
     3961 ttttaacaac ataatatgct attgcatttc tgggaaattc tgggtaaatt ccaagcatgc
     4021 agcaattagt ttgtgcttat gtgtgaagtc cagttaggtt ctcagttcat accctgaaaa
     4081 ctgcttattc cacatctgtt gatgtggtcc actgggggca gttctgttcc tcaccatctt
     4141 ggcagcatgc atggctcctg cccacatttc aaccacaccc ccaacccaat tacgtgacca
     4201 gcccccacga aatttcacag cattccgtgc aggggagtgt cacagctcct cttgaaaaac
     4261 atctggtgtt gctatgagcc agaagggtac cgagctcgaa ttcactggcc gtcgttttac
     4321 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     4381 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     4441 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4501 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4561 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4621 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4681 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4741 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4801 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4861 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4921 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4981 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5041 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5101 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5161 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5221 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5281 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5341 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5401 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5461 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5521 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5581 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     5641 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5701 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5761 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5821 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5881 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5941 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6001 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6061 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6121 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6181 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6241 cgggctgaac gggggggtcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6301 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6361 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6421 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6481 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6541 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     6601 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     6661 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     6721 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     6781 aagcgggcag tgagcgcaac gcaat
//
