LOCUS       sequence-211688-        7158 bp    DNA     circular SYN 15-APR-2025
DEFINITION  CRISPR donor plasmid to tag human transcription factor HEY1 with 
            GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7158)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 7158)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pHEY1-donor
FEATURES             Location/Qualifiers
     source          1..7158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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(join(82..88,89..106,107..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(join(1195..1986,1987..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             3894..3914
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3927..3971
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             4011..4034
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(4062..4064,4065..4067,4068..4781)
                     /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(4107..4128)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4368..4387)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(4819..5392,5393..5395,5396..5404)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4985..5002)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5212..5231
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5486..6289
                     /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(5549..5568)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6159..6178
                     /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 tagaaggaca 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 tcggtaccct gtgacagccc tgattcagat caggtagctt
     2701 tcacatttgt ccattgctat tttgagaaaa agcatttaaa aacaatatgt ggtcttattt
     2761 ttggtatacg ggattttaaa aaaaaggtgc tgccacatgc ctcttgagga aagaaatgta
     2821 ttctggttaa gtaaaggaaa cctaacggga gaccaaggtc tgtgtgtatg tctctccagc
     2881 aggaaaatgc tttactgagg tccaagtgcc taaaagctcc gcgatgataa cttacatctg
     2941 tgtgtctaat gaatttcact gctctgtggt ttctctgccc ttgatgtatt ttttgttgtt
     3001 gttgagggaa aacattaaag gaatgaacat agtttcatct gctggtaggg tataattcca
     3061 atttctgctc tgcagcttct aattgggctt tgaacttgca ttagcaaaca cattctgctt
     3121 atgttgatta atgcaaagat ttttgttgat tccaaagact ttcggaagaa cccctgactg
     3181 ttgtgtttta agggatgcag gtatctgcca ggaagggctg tctctgggac ttaagaaatg
     3241 agggaaacgg gcaaagccct gagatttttc ttttgttctt ttgcttaggt tactttgacg
     3301 cgcacgccct tgctatggac tatcggagtt tgggatttcg ggaatgcctg gcagaagttg
     3361 cgcgttatct gagcatcatt gaaggactag atgcctctga cccgcttcga gttcgactgg
     3421 tttcgcatct caacaactac gcttcccagc gggaagccgc gagcggcgcc cacgcgggcc
     3481 tcggacacat tccctggggg accgtcttcg gacatcaccc gcacatcgcg cacccgctgt
     3541 tgctgcccca gaacggccac gggaacgcgg gcaccacggc ctcacccacg gaaccgcacc
     3601 accagggcag gctgggctcg gcacatccgg aggcgcctgc tttgcgagcg ccccctagcg
     3661 gcagcctcgg accggtgctc cctgtggtca cctccgcctc caaactgtcg ccgcctctgc
     3721 tctcctcagt ggcctccctg tcggccttcc ccttctcttt cggctccttc cacttactgt
     3781 ctcccaatgc actgagccct tcagcaccca cgcaggctgc aaaccttggc aagccctata
     3841 gaccttgggg gacggagatc ggagcttttc ccgggattcc aactactgca agcgagaatc
     3901 tttattttca gggcgccgcc aaattcaaag aaaccgctgc tgctaaattc gaacgccagc
     3961 acatggacag cggaggtgga ggttcgagcg gtccctcggg ttcgtcgagc ctggaagttc
     4021 tgttccaggg gcccctgtcg tcgagcggtc cctcgggttc gatggtgagc aaaggcgagg
     4081 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     4141 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     4201 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     4261 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     4321 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     4381 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     4441 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     4501 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     4561 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     4621 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     4681 ccctgagcaa agaccccaac gagaagcgcg atcacatggt ccttaaggag ttcgtgaccg
     4741 ccgccgggat cactctcggc atggacgagc tgtacaagta atgatctaga gtcgagttaa
     4801 ttaagaattc cgcccccccc cctctccctc ccccccccta acgttactgg ccgaagccgc
     4861 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     4921 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     4981 tcccctctcg ccaaaggaat gcagggtctg ttgaatgtcg tgaaggaagc agttcctctg
     5041 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     5101 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     5161 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     5221 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     5281 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
     5341 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac
     5401 aacctgttac attgcacaag ataaaaatat atcatcacga acagtaaaac tgtctgctta
     5461 cataaacagt aatacaaggg gtgttatggg atcggccatt gaacaagatg gattgcacgc
     5521 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     5581 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     5641 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     5701 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     5761 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     5821 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     5881 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     5941 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     6001 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     6061 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     6121 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     6181 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     6241 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa gaactgatgt
     6301 agaatgaggg aggggaaagt ttaaaatccc agctgggctg gactgttgcc aacatcacct
     6361 taaagtcgtc agtaaaagta aaaaggaaaa aggtacactt tcagataatt ttttttttaa
     6421 agactaaagg tttgttggtt tacttttatc ttttttaatg tttttttcat catgtcatgt
     6481 attagcagtt tttaaaaact agttgttaaa ttttgttcaa gacattaaat tgaaatagtg
     6541 agtataagcc aacactttgt gataggtttg tactgtgcct aatttacttt gtaaaccaga
     6601 atgattccgt ttttgcctca aaatttgggg aatcttaaca tttagtattt ttggtctgtt
     6661 tttctccttg tatagttatg gtctgttttt agaattaatt ttccaaacca ctatgcttaa
     6721 tgttaacatg attctgtttg ttaatatttt gacagattaa ggtgttgtat aaataatatt
     6781 cttttggggg gaggggaact atattgaatt ttatatttct gagcaaagcg ttgacaaatc
     6841 agatgatcag ctttatccaa gaaagaagac tagtaaattg tctgcctcct atagcagaaa
     6901 ggtgaatgta caaactgttg gtggccctga atccatctga ccagctgctg gtatctgcca
     6961 ggactggcag ttctgattta gttaggagag agccgctgat aggttaggtc tcatttggag
     7021 tgttggtgga aaggaaactg aaggtaattg aatagaatac gcctgcattt accagcccca
     7081 gcaacacaaa gaatttttaa tcacacggat ctcaaattca caaatgttaa catggataag
     7141 tgatcatggt gtgcgagt
//
