LOCUS       Exported                7128 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR donor plasmid to tag human transcription factor E2F5 with 
            GFP.
ACCESSION   .
VERSION     .
KEYWORDS    pE2F5-donor
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7128)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 7128)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7128
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     promoter        complement(82..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(1195..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             3645..3665
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3678..3722
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3762..3785
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             3813..4532
                     /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(3858..3879)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4119..4138)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    4570..5155
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4736..4753)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4963..4982
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5237..6040
                     /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(5300..5319)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5910..5929
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gacctgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
       61 ttatccgttc 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 tctccctctg tcatccaggc tggagtgtca
     2701 tccaggctgg agtgcagtgg catgatagct cactgcaacc tccacctcct aggttcaaat
     2761 gattctcctg gctcagcctc ccgagtagct gggatgacag gcacctgcca ccacacctgg
     2821 ctactttttg tatttttagt agagacgggg tttcaccatg ttggccaggc tggtctcaaa
     2881 ctcctggcct caggtgatcc acctgcctca gcctcccaaa gtgttgggat tacaggcatg
     2941 agccactgca cctggcctcg ttagtgcctt ttagttatat taccttagtg ataagttcaa
     3001 ccaataaaaa atttaatttc taggccgggc acaatggctc atgcctgtaa tcccagcact
     3061 ttgggaggcc aaggcgggcg aatcacgtca ggagatcgag accatcctgg ctaacatggt
     3121 gaaaccccat ctctataaaa aaataaaaaa aatgagccgg tcgtggtggc aggcacctgt
     3181 agtcccagct actcgggagg ctgaggcagg agaatggcgt gaacccggga gtcgggagct
     3241 tgcagtgggc cgagatcgag ctactgcact ccagcctgga cgaaacaggg agactccatc
     3301 ttaaaaaaaa aaaaaaaaaa aaaaagaaaa aattaacttc tgtttctcaa tctaatatca
     3361 atattcaatg tacaaattgt tactaaatga gaatatttca aaatacacat tacacaagac
     3421 tttaatttgg aatgatgtat ttgagagtat tttgttaaag atcattaacc gtatgtagaa
     3481 accatcttta acactaaatt tgttttttgt tcgcagtgtt tcctctctta aggctttctc
     3541 ctaccccggc agatgactac aactttaatt tagatgataa cgaaggagtt tgtgatctgt
     3601 ttgatgtcca gatactaaat tacgggattc caactactgc aagcgagaat ctttattttc
     3661 agggcgccgc caaattcaaa gaaaccgctg ctgctaaatt cgaacgccag cacatggaca
     3721 gcggaggtgg aggttcgagc ggtccctcgg gttcgtcgag cctggaagtt ctgttccagg
     3781 ggcccctgtc gtcgagcggt ccctcgggtt cgatggtgag caaaggcgag gagctgttca
     3841 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     3901 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     3961 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     4021 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     4081 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     4141 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     4201 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     4261 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     4321 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     4381 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     4441 aagaccccaa cgagaagcgc gatcacatgg tccttaagga gttcgtgacc gccgccggga
     4501 tcactctcgg catggacgag ctgtacaagt aatgatctag agtcgagtta attaagaatt
     4561 ccgccccccc ccctctccct ccccccccct aacgttactg gccgaagccg cttggaataa
     4621 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg
     4681 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc
     4741 gccaaaggaa tgcagggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct
     4801 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac
     4861 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc
     4921 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta
     4981 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg
     5041 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg
     5101 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca caacctgtta
     5161 cattgcacaa gataaaaata tatcatcacg aacagtaaaa ctgtctgctt acataaacag
     5221 taatacaagg ggtgttatgg gatcggccat tgaacaagat ggattgcacg caggttctcc
     5281 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     5341 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     5401 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     5461 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     5521 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     5581 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     5641 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     5701 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     5761 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     5821 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     5881 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     5941 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     6001 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gtctagattc catggaaact
     6061 tgggactgtt atctacctct aactgtgtaa cattttagac ttcttaataa cctaaatatt
     6121 taaaataatg aatgtaacac cttttttagt tcactgattc tgaagtgttc ttccctaata
     6181 ctttctttac ttcacaaaac ttcaaccata aaaacaaagg gctctgattg ctttagggga
     6241 taagtgattt aatatccaca aacgtcccca ctcccaaaag taactatatt ctggatttca
     6301 acttttcttc taattgtgaa tccttctgtt ttttcttctt aaggaggaaa gttaaaggac
     6361 actacaggtc atcaaaaaca agttggccaa ggactcatta cttgtcttat atttttactg
     6421 ccactaaact gcctgtattt ctgtatgtcc ttctatccaa acagacgttc actgccactt
     6481 gtaaagtgaa ggatgtaaac gaggatatat aactgtttca gtgaacagat tttgtgaagt
     6541 gccttctgtt ttagcacttt aagtttatca cattttgttg acttctgaca ttccactttc
     6601 ctaggttata ggaaagatct gtttatgtag tttgttttta aaatgtgcca atgcctgtac
     6661 attaacaaga tttttaaaaa taaaattgta taaaacattc aatttattgg tctttgtgga
     6721 gaattagatg catcaccagt atattacaac agagccatta atcttgtagc ttcatcaaca
     6781 ttaactggtt tgctttcatg acgctgctga ggaatctgaa aggagaaagt attatattta
     6841 aaaacacaga caacaatagc agacaaatga cttatataag ctgttattca atttaatttt
     6901 tctagtcata tctttggaag tagaacaatc tgtatattct gacaatgttt tagagtctga
     6961 atatgctatt tatactatag agcctagtac tagagagcca agtactaaaa attacactca
     7021 acttacaggc tttgaaaact tgggagttta atttattagc ctcaatttgt tagccataaa
     7081 aaaaagcaaa tgatttctgc ttaccagttc tttctgcaga ggttcaag
//
