LOCUS       sequence-214768-        7956 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 7956)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 7956)
  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..7956
                     /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      1896..1905
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1902..2276
                     /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      2414..3002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2903..2922
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3156..3173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3290..3311
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3326..3331,3332..3349,3350..3356)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3364..3380
                     /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     3369..3391
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3388..3404
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3388..3404
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3422..3440
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3422..3439
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             3964..3981
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4701..4721
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4734..4778
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             4818..4841
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(4869..4871,4872..4874,4875..5588)
                     /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(4914..4935)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5175..5194)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(5626..6199,6200..6202,6203..6211)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5792..5809)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     6019..6038
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             6293..7096
                     /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"
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 ctcgccgcca
      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 ccgcatacag gtggcacttt tcggggaaat gtgcgcggaa
     1801 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1861 cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag ttgaccagtg
     1921 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     1981 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     2041 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     2101 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     2161 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     2221 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     2281 tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     2341 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2401 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2461 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2521 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     2581 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2641 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2701 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2761 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2821 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2881 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2941 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3001 aaaacgccag caacgcggcc tttttacggt tcctgggctt ttgctggcct tttgctcaca
     3061 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3121 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3181 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3241 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     3301 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     3361 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     3421 tatttaggtg acactataga atactcaagc tatgcatcaa gcttggtacc gagctcggat
     3481 ccactagtaa cggccgccag tgtgctggaa ttcgcccttt tgcccaggta gaatctgctc
     3541 ccatgcaggt ggtccagaag gagcctgttc agatggagct gtctcctccc atggaggtgg
     3601 tccagaagga gcctgttcag atagagctgt ctcctcccat ggaggtggtc cagaaggaac
     3661 ctgttaagat agagctgtct cctcccatag aggtggtcca gaaggagcct gttcagatgg
     3721 agttgtctcc tcccatgggg gtggttcaga aggagcctgc tcagagggag ccacctcctc
     3781 ccagagagcc tccccttcac atggagccaa tttccaaaaa gcctcctctc cgaaaagata
     3841 aaaaggaaaa gtctaacatg cagagtgaaa gggcacggaa ggagcaagtc cttattgaag
     3901 ttggcttagt gcctgttaaa gatagctggc ttctaaagga aagtgtaagc acagaggatc
     3961 tctcaccacc atcaccacca ctgccaaagg aaaatttaag agaagaggca tcaggagacc
     4021 aaaaattact caacacaggt gaaggaaata aagaagcccc tcttcagaaa gtaggagcag
     4081 aagaggcaga tgagagccta cctggtcttg ctgctaatat caacgaatct acccatattt
     4141 catcctctgg acaaaacttg aatacgccag agggtgaaac tttaaatggt aaacatcaga
     4201 ctgacagtat agtttgtgaa atgaaaatgg acactgatca gaacacaaga gagaatctca
     4261 ctggtataaa ttcaacagtt gaagaaccag tttcaccaat gcttccccct tcagcagtag
     4321 aagaacgtga agcagtgtcc aaaactgcac tggcatcacc tcctgctaca atggcagcaa
     4381 atgagtctca ggaaattgat gaagatgaag gcatccacag ccatgaagga agtgacctaa
     4441 gtgacaacat gtcagagggt agtgatgatt ctggattgca tggggctcgg ccagttccac
     4501 aagaatctag cagaaaaaat gcaaaggaag ccttggcagt caaagcggct aagggagatt
     4561 ttgtttgtat cttctgtgat cgttctttca gaaagggaaa agattacagc aaacacctca
     4621 atcgccattt ggttaatgtg tactatcttg aagaagcagc tcaagggcag gaggattatg
     4681 atattccaac tactgcaagc gagaatcttt attttcaggg cgccgccaaa ttcaaagaaa
     4741 ccgctgctgc taaattcgaa cgccagcaca tggacagcgg aggtggaggt tcgagcggtc
     4801 cctcgggttc gtcgagcctg gaagttctgt tccaggggcc cctgtcgtcg agcggtccct
     4861 cgggttcgat ggtgagcaaa ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     4921 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     4981 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     5041 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     5101 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     5161 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     5221 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     5281 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     5341 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     5401 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     5461 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     5521 acatggtcct taaggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     5581 acaagtaatg atctagagtc gagttaatta agaattccgc ccccccccct ctccctcccc
     5641 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     5701 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     5761 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca gggtctgttg
     5821 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     5881 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     5941 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     6001 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     6061 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     6121 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     6181 tgaaaaacac gatgataata tggccacaac ctgttacatt gcacaagata aaaatatatc
     6241 atcacgaaca gtaaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgggatc
     6301 ggccattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     6361 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     6421 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     6481 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     6541 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     6601 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     6661 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     6721 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     6781 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     6841 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     6901 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     6961 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     7021 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     7081 tgacgagttc ttctgatgaa actttgaaca aggtttcagt tcttagtttg taaggtatat
     7141 tacattttat attcatttat gatagcagac aaccttttaa gattgcttta attagtatct
     7201 gatgttgatt tttaagtggc attcttttcc ttaggacttt ttatgtatac ctgttgattg
     7261 ttgtgtaaat tttagtaaat ctaagagagt gtactaaacc agcaggtatc tgttagctta
     7321 tgtgtttaat tgaaattaga aggctaagat ggtataacag cattttattg ctttgtccag
     7381 ctacaacttg tcattttttt ctccatgtct tatcttcctg tttcacttta gtttattctt
     7441 cgttttttat tgagatctat aaaaaattgg cttacttaat agcaaattac ttgaagaatt
     7501 tgcctgcttt atataaagtt agcactttaa gatttttttt tttagagatg agaagacatt
     7561 taaattgaag aaaaattccc ccagcaatag acagtctatc agtccaagta tttacttcct
     7621 gagttttgat caatattttt tatttgtgta tgttaatcgt cataaaaaca gtgattttgg
     7681 tgtgtttttt attttggtgc tttaatggct taagatgttg cacatttttt ttttcttttg
     7741 gtttctgttt atgttttttt gcctatgcag ttaaattttt cctagaaata gcatttgtgt
     7801 tgaacagtaa cactttatac atatatatat gcatgtttat tttgtttggc gtctttggag
     7861 ggatgctttt agacttgttt gcaaaagggc agttttcttt ttctttgctg cagttgtcta
     7921 ttttgcagaa taatagtgtg tgcaagtttg tgagca
//
