LOCUS       Exported                7159 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPR donor plasmid to tag human transcription factor HEY1 with 
            GFP.
ACCESSION   .
VERSION     .
KEYWORDS    pHEY1-donor
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7159)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 7159)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7159
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1259..1279
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1292..1336
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1376..1399
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1427..2146
                     /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(1472..1493)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1733..1752)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    2184..2769
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2350..2367)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2577..2596
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2851..3654
                     /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(2914..2933)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3524..3543
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4560..4576)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4560..4576)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4573..4595)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4584..4600
                     /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(4608..4638)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4653..4674
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4791..4808)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4962..5550)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5042..5061)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5721..6581)
                     /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     6344..6363
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6582..6686)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6754..6772
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6810..6832)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6932..6951
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7145..8
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7159..17
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttcgagctcg gtaccctgtg acagccctga ttcagatcag
       61 gtagctttca catttgtcca ttgctatttt gagaaaaagc atttaaaaac aatatgtggt
      121 cttatttttg gtatacggga ttttaaaaaa aaggtgctgc cacatgcctc ttgaggaaag
      181 aaatgtattc tggttaagta aaggaaacct aacgggagac caaggtctgt gtgtatgtct
      241 ctccagcagg aaaatgcttt actgaggtcc aagtgcctaa aagctccgcg atgataactt
      301 acatctgtgt gtctaatgaa tttcactgct ctgtggtttc tctgcccttg atgtattttt
      361 ttgttgttgt tgagggaaaa cattaaagga atgaacatag tttcatctgc tggtagggta
      421 taattccaat ttctgctctg cagcttctaa ttgggctttg aacttgcatt agcaaacaca
      481 ttctgcttat gttgattaat gcaaagattt ttgttgattc caaagacttt cggaagaacc
      541 cctgactgtt gtgttttaag ggatgcaggt atctgccagg aagggctgtc tctgggactt
      601 aagaaatgag ggaaacgggc aaagccctga gatttttctt ttgttctttt gcttaggtta
      661 ctttgacgcg cacgcccttg ctatggacta tcggagtttg ggatttcggg aatgcctggc
      721 agaagttgcg cgttatctga gcatcattga aggactagat gcctctgacc cgcttcgagt
      781 tcgactggtt tcgcatctca acaactacgc ttcccagcgg gaagccgcga gcggcgccca
      841 cgcgggcctc ggacacattc cctgggggac cgtcttcgga catcacccgc acatcgcgca
      901 cccgctgttg ctgccccaga acggccacgg gaacgcgggc accacggcct cacccacgga
      961 accgcaccac cagggcaggc tgggctcggc acatccggag gcgcctgctt tgcgagcgcc
     1021 ccctagcggc agcctcggac cggtgctccc tgtggtcacc tccgcctcca aactgtcgcc
     1081 gcctctgctc tcctcagtgg cctccctgtc ggccttcccc ttctctttcg gctccttcca
     1141 cttactgtct cccaatgcac tgagcccttc agcacccacg caggctgcaa accttggcaa
     1201 gccctataga ccttggggga cggagatcgg agcttttggg attccaacta ctgcaagcga
     1261 gaatctttat tttcagggcg ccgccaaatt caaagaaacc gctgctgcta aattcgaacg
     1321 ccagcacatg gacagcggag gtggaggttc gagcggtccc tcgggttcgt cgagcctgga
     1381 agttctgttc caggggcccc tgtcgtcgag cggtccctcg ggttcgatgg tgagcaaagg
     1441 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1501 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     1561 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1621 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1681 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1741 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1801 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1861 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1921 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1981 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     2041 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctta aggagttcgt
     2101 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaatgat ctagagtcga
     2161 gttaattaag aattccgccc ccccccctct ccctcccccc ccctaacgtt actggccgaa
     2221 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
     2281 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg
     2341 gtctttcccc tctcgccaaa ggaatgcagg gtctgttgaa tgtcgtgaag gaagcagttc
     2401 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc
     2461 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
     2521 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
     2581 tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg
     2641 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac
     2701 gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataatatg
     2761 gccacaacct gttacattgc acaagataaa aatatatcat cacgaacagt aaaactgtct
     2821 gcttacataa acagtaatac aaggggtgtt atgggatcgg ccattgaaca agatggattg
     2881 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     2941 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3001 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3061 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3121 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3181 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3241 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3301 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3361 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     3421 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3481 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3541 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     3601 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagtcaga
     3661 actgatgtag aatgagggag gggaaagttt aaaatcccag ctgggctgga ctgttgccaa
     3721 catcacctta aagtcgtcag taaaagtaaa aaggaaaaag gtacactttc agataatttt
     3781 ttttttaaag actaaaggtt tgttggttta cttttatctt ttttaatgtt tttttcatca
     3841 tgtcatgtat tagcagtttt taaaaactag ttgttaaatt ttgttcaaga cattaaattg
     3901 aaatagtgag tataagccaa cactttgtga taggtttgta ctgtgcctaa tttactttgt
     3961 aaaccagaat gattccgttt ttgcctcaaa atttggggaa tcttaacatt tagtattttt
     4021 ggtctgtttt tctccttgta tagttatggt ctgtttttag aattaatttt ccaaaccact
     4081 atgcttaatg ttaacatgat tctgtttgtt aatattttga cagattaagg tgttgtataa
     4141 ataatattct tttgggggga ggggaactat attgaatttt atatttctga gcaaagcgtt
     4201 gacaaatcag atgatcagct ttatccaaga aagaagacta gtaaattgtc tgcctcctat
     4261 agcagaaagg tgaatgtaca aactgttggt ggccctgaat ccatctgacc agctgctggt
     4321 atctgccagg actggcagtt ctgatttagt taggagagag ccgctgatag gttaggtctc
     4381 atttggagtg ttggtggaaa ggaaactgaa ggtaattgaa tagaatacgc ctgcatttac
     4441 cagccccagc aacacaaaga atttttaatc acacggatct caaattcaca aatgttaaca
     4501 tggataagtg atcatggtgt gcgagtgacc tgcaggcatg caagcttggc gtaatcatgg
     4561 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4621 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4681 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4741 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4801 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4861 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4921 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4981 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5041 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5101 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5161 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5221 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5281 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5341 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5401 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5461 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5521 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5581 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5641 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5701 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5761 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5821 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5881 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5941 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6001 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6061 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6121 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6181 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6241 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6301 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6361 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6421 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6481 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6541 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6601 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6661 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     6721 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     6781 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     6841 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     6901 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
     6961 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     7021 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     7081 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     7141 ttttcccagt cacgacgtt
//
