LOCUS       Exported                7104 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR donor plasmid to tag human transcription factor EHF with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    pEHF-donor
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7104)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 7104)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7104
                     /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"
     primer_bind     complement(47..69)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    58..74
                     /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)."
     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             3879..3899
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3912..3956
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3996..4019
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4047..4766
                     /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(4092..4113)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4353..4372)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    4804..5389
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4970..4987)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5197..5216
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5471..6274
                     /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(5534..5553)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6144..6163
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gacctgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
       61 ttatccgctc 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 tcggtaccca attgcaccac tgtactccag cttgggccac
     2701 agagtgatat cctgtctcag aaaaaaaaaa aaggtataat ccagaaatta tgaggaatta
     2761 gaatgtcttt atctgctctt tcacataatc ccacaaaacc atattaatgc aacccctcga
     2821 aaggtgggtg gggtgggggt ggggcatcaa aaattcagtg ggagtaaagt tgaatttggt
     2881 tgataatctt attgcagtag tggcttcatt ttcaaattgg gtcctccagt ccctgcgtga
     2941 ccaccccttt ccctaccacc ctcatctatt ctgggagtaa acattctaga cccttactga
     3001 tttgcatttg gctgaaacac agttgctggg tcaaagcaat ttaattggtt gagatactgg
     3061 ataaaagtgg ttgagagttg ctctctgggg aagtaacagg ttgaagtgaa ccccaagcaa
     3121 tttgtggata ataactagaa gaaacttccc cctggcttag gagacctaag acctattgct
     3181 tagtcttggc tatgtcacta gttagctgtg tgatcctgat caagtcactt aagttctctg
     3241 tgcctcggtt tcctgatttg taaaataaaa tattgtgcta gtttattctt aaataccttt
     3301 ttaatgtcca acatctggtg attctgcgat ccatcttccc tgacaagtag gggagaaatg
     3361 agtatctcct acttgcagga agatgaccta actcaatgct ctctgcccta tctttgctgt
     3421 gacttagatc attaataacc tgcctttctg cttttcatca gacccgagag ggactcactt
     3481 atgggaattc atccgcgaca tcctcttgaa cccagacaag aacccaggat taataaaatg
     3541 ggaagaccga tctgagggcg tcttcaggtt cttgaaatca gaggcagtgg ctcagctatg
     3601 gggtaaaaag aagaacaaca gcagcatgac ctatgaaaag ctcagccgag ctatgaggtg
     3661 aggagtttca tgtctctgaa aacaaaaagg ctgtacgacc cattatttac ctagcaacct
     3721 tcatcggatc agtcacctta tgcaatctcc tttgttacag atattactac aaaagagaaa
     3781 ttctggagcg tgtggatgga cgaagactgg tatataaatt tgggaagaat gcccgaggat
     3841 ggagagaaaa tgaaaacggg attccaacta ctgcaagcga gaatctttat tttcagggcg
     3901 ccgccaaatt caaagaaacc gctgctgcta aattcgaacg ccagcacatg gacagcggag
     3961 gtggaggttc gagcggtccc tcgggttcgt cgagcctgga agttctgttc caggggcccc
     4021 tgtcgtcgag cggtccctcg ggttcgatgg tgagcaaagg cgaggagctg ttcaccgggg
     4081 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     4141 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     4201 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     4261 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     4321 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     4381 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     4441 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     4501 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     4561 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     4621 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     4681 ccaacgagaa gcgcgatcac atggtcctta aggagttcgt gaccgccgcc gggatcactc
     4741 tcggcatgga cgagctgtac aagtaatgat ctagagtcga gttaattaag aattccgccc
     4801 ccccccctct ccctcccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg
     4861 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc
     4921 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa
     4981 ggaatgcagg gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
     5041 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
     5101 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
     5161 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
     5221 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
     5281 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
     5341 ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacct gttacattgc
     5401 acaagataaa aatatatcat cacgaacagt aaaactgtct gcttacataa acagtaatac
     5461 aaggggtgtt atgggatcgg ccattgaaca agatggattg cacgcaggtt ctccggccgc
     5521 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     5581 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     5641 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     5701 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     5761 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     5821 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     5881 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     5941 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     6001 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     6061 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     6121 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     6181 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     6241 cgccttctat cgccttcttg acgagttctt ctgagtcagc tgccaatact ttggacacaa
     6301 accaaaacac acaccaaata atcagaaaca aagaactcct ggacgtaaat atttcaaaga
     6361 ctacttttct ctgatattta tgtaccatga ggggaaaaag aaactacttc taacgggaag
     6421 aagaaacact acagtcgatt aaaaaaatta ttttgttact tcgaagtatg tcctatatgg
     6481 ggaaaaaacg tacacagttt tctgtgaaat atgatgctgt atgtggttgt gatttttttt
     6541 cacctctatt gtgaattctt tttcactgca agagtaacag gatttgtagc cttgtgcttc
     6601 ttgctaagag aaagaaaaac aaaatcagag ggcattaaat gttttgtatg tgacatgatt
     6661 tagaaaaagg tgatgcatcc tcctcacata agcatccata tggcttcgtc aagggaggtg
     6721 aacattgttg ctgagttaaa ttccagggtc tcagatggtt aggacaaagt ggatggatgc
     6781 cgggaagttt aacctgagcc ttaggatcca atgagtggag aatggggact tccaaaaccc
     6841 aaggttggct ataatctctg cataaccaca tgacttggaa tgcttaaatc agcaagaaga
     6901 ataatggtgg ggtctttata ctcattcagg aatggtttat ctgatgccag ggctgtcttc
     6961 ctttctcccc tttggatggt tggtgaaata ctttaattgc cctgtctgct cacttctagc
     7021 tatttaagag agaacccagc ttggttcttt tttgctccaa gtgcttaaaa ataagttgga
     7081 aaaaggagac ggtggtgtgg aaat
//
