LOCUS       sequence-231037-        7967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR donor plasmid to tag human transcription factor REST with 
            GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7967)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 7967)
  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: pREST-donor
FEATURES             Location/Qualifiers
     source          1..7967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(70..89)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(71..89)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(96..113)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(96..112)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(105..127)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             250..552
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     431..450
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             901..1695
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             901..1695
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     regulatory      1895..1904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1901..2275
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      2413..3001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2902..2921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3155..3172
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3289..3310
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3325..3330,3331..3348,3349..3355)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3363..3379
                     /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     3368..3390
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3387..3403
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3387..3403
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3421..3439
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3421..3438
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             3973..3990
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4710..4730
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4743..4787
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             4827..4850
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(4878..4880,4881..4883,4884..5597)
                     /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(4923..4944)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5184..5203)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(5635..6208,6209..6211,6212..6220)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5801..5818)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     6028..6047
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             6302..7105
                     /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="MGSTIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
ORIGIN
        1 aagggcgaat tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg
       61 cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt
      121 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
      181 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagccta
      241 tacgtacggc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
      301 gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc cctggccagt
      361 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat
      421 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa
      481 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
      541 tggggaatat aaatgtcagg catgagatta tcaaaaagga tcttcaccta gatccttttc
      601 acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct
      661 atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca
      721 tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg
      781 gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca
      841 aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg atcgtttcgc
      901 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
      961 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     1021 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     1081 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     1141 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     1201 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     1261 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     1321 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     1381 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     1441 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     1501 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     1561 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     1621 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     1681 gacgagttct tctgaattat taacgcttac aatttcctga tgcggtattt tctccttacg
     1741 catctgtgcg gtatttcaca ccgcatcagg tggcactttt cggggaaatg tgcgcggaac
     1801 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1861 ctgataaatg cttcaataat agcacgtgag gagggccacc atggccaagt tgaccagtgc
     1921 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
     1981 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
     2041 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
     2101 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
     2161 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
     2221 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt
     2281 gctaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     2341 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2401 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2461 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2521 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2581 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2641 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2701 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2761 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2821 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2881 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2941 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3001 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3061 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3121 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3181 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3241 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     3301 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     3361 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     3421 atttaggtga cactatagaa tactcaagct atgcatcaag cttggtaccg agctcggatc
     3481 cactagtaac ggccgccagt gtgatggata tctgcagaat tcgccctttt gcccaggtag
     3541 aatctgctcc catgcaggtg gtccagaagg agcctgttca gatggagctg tctcctccca
     3601 tggaggtggt ccagaaggag cctgttcaga tagagctgtc tcctcccatg gaggtggtcc
     3661 agaaggaacc tgttaagata gagctgtctc ctcccataga ggtggtccag aaggagcctg
     3721 ttcagatgga gttgtctcct cccatggggg tggttcagaa ggagcctgct cagagggagc
     3781 cacctcctcc cagagagcct ccccttcaca tggagccaat ttccaaaaag cctcctctcc
     3841 gaaaagataa aaaggaaaag tctaacatgc agagtgaaag ggcacggaag gagcaagtcc
     3901 ttattgaagt tggcttagtg cctgttaaag atagctggct tctaaaggaa agtgtaagca
     3961 cagaggatct ctcaccacca tcaccaccac tgccaaagga aaatttaaga gaagaggcat
     4021 caggagacca aaaattactc aacacaggtg aaggaaataa agaagcccct cttcagaaag
     4081 taggagcaga agaggcagat gagagcctac ctggtcttgc tgctaatatc aacgaatcta
     4141 cccatatttc atcctctgga caaaacttga atacgccaga gggtgaaact ttaaatggta
     4201 aacatcagac tgacagtata gtttgtgaaa tgaaaatgga cactgatcag aacacaagag
     4261 agaatctcac tggtataaat tcaacagttg aagaaccagt ttcaccaatg cttccccctt
     4321 cagcagtaga agaacgtgaa gcagtgtcca aaactgcact ggcatcacct cctgctacaa
     4381 tggcagcaaa tgagtctcag gaaattgatg aagatgaagg catccacagc catgaaggaa
     4441 gtgacctaag tgacaacatg tcagagggta gtgatgattc tggattgcat ggggctcggc
     4501 cagttccaca agaatctagc agaaaaaatg caaaggaagc cttggcagtc aaagcggcta
     4561 agggagattt tgtttgtatc ttctgtgatc gttctttcag aaagggaaaa gattacagca
     4621 aacacctcaa tcgccatttg gttaatgtgt actatcttga agaagcagct caagggcagg
     4681 aggattatga tattccaact actgcaagcg agaatcttta ttttcagggc gccgccaaat
     4741 tcaaagaaac cgctgctgct aaattcgaac gccagcacat ggacagcgga ggtggaggtt
     4801 cgagcggtcc ctcgggttcg tcgagcctgg aagttctgtt ccaggggccc ctgtcgtcga
     4861 gcggtccctc gggttcgatg gtgagcaaag gcgaggagct gttcaccggg gtggtgccca
     4921 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     4981 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     5041 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     5101 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     5161 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     5221 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     5281 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     5341 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     5401 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     5461 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     5521 agcgcgatca catggtcctt aaggagttcg tgaccgccgc cgggatcact ctcggcatgg
     5581 acgagctgta caagtaatga tctagagtcg agttaattaa gaattccgcc cccccccctc
     5641 tccctccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt
     5701 gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct
     5761 ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcag
     5821 ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg
     5881 tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc
     5941 caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg
     6001 agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg
     6061 aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc
     6121 tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg
     6181 gttttccttt gaaaaacacg atgataatat ggccacaacc tgttacattg cacaagataa
     6241 aaatatatca tcacgaacag taaaactgtc tgcttacata aacagtaata caaggggtgt
     6301 tatgggatcg accattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     6361 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     6421 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     6481 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     6541 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     6601 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     6661 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     6721 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     6781 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     6841 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     6901 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     6961 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     7021 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     7081 tcgccttctt gacgagttct tctgataatg aaactttgaa caaggtttca gttcttagtt
     7141 tgtaaggtat attacatttt atattcattt atgatagcag acaacctttt aagattgctt
     7201 taattagtat ctgatgttga tttttaagtg gcattctttt ccttaggact ttttatgtat
     7261 acctgttgat tgttgtgtaa attttagtaa atctaagaga gtgtactaaa ccagcaggta
     7321 tctgttagct tatgtgttta attgaaatta gaaggctaag atggtataac agcattttat
     7381 tgctttgtcc agctacaaca tgtcattttt ttctccatgt cttatcttcc tgtttcactt
     7441 tagtttattc ttcgtttttt attgagatct ataaaaaatt ggcttactta atagcaaatt
     7501 acttgaagaa tttgcctgct ttatataaag ttagcacttt aagatttttt ttttagagat
     7561 gagaagacat ttaaattgaa gaaaaattcc cccagcaata gacagtctat cagtccaagt
     7621 atttacttcc tgagttttga tcaatatttt ttatttgtgt atgttaatcg tcataaaaac
     7681 agtgattttg gtgtgttttt tattttggtg ctttaatggc ttaagatgtt gcacattttt
     7741 tttttctttt ggtttctgtt tatgtttttt tgcctatgca gttaaatttt tcctagaaat
     7801 agcatttgtg ttgaacagta acactttata catatatata tgcatgttta ttttgtttgg
     7861 cgtctttgga gggatgcttt tagacttgtt tgcaaaaggg cagttttctt tttctttgct
     7921 gcagttgtct attttgcaga ataatagtgt gtgcaagttt gtgagca
//
