LOCUS       sequence-273771-        7341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR donor plasmid to create GFP fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7341)
  TITLE     ENCODE collection: CRISPR constructs
REFERENCE   2  (bases 1 to 7341)
  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: pTP63-donor
FEATURES             Location/Qualifiers
     source          1..7341
                     /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             3852..3872
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3885..3929
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3969..3992
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(4020..4022,4023..4025,4026..4739)
                     /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(4065..4086)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4326..4345)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(4777..5350,5351..5353,5354..5362)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4943..4960)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5170..5189
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5444..6247
                     /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(5507..5526)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6117..6136
                     /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 tcggtacccg tgaccatcaa aacctctgcc acagttttct
     2701 cactccaagc acaatgtgct gtttacctac cattctgccc tgtgaaaata tgagctatca
     2761 ctgaaacaat ctaaagaaga gctaacgcag ataaatgtaa gtgatatcac cacagtcagt
     2821 taaggttgac cctatttgca atgccttctg cacccattca cagaacatag acacagttgc
     2881 tctgcatttt gataccttgc tttattcaaa caccaagaga ccttttagat aaaagtgccg
     2941 tttcaggttc tgaggatgcc ctaagtccct aattttttca cttagttatg tgactttaaa
     3001 cttgtcaacc tctctggcct tcagttgttc tatctataaa acaggtctaa taatgtttac
     3061 cactaatctg gggattaaat gatatcatga atataaaata attcagggcc tggcacatag
     3121 aaagtactca atcttttctt ctcatctcct caaccaagtg gtggtgacat tccattaata
     3181 cctttcttct agtgactagc caggtaaatt caagcataag taggtacctc atgtttctat
     3241 ttgggatttt tgccctctca tctagctatt atcccaattt taccaatgaa gaaactgagg
     3301 ccagtagaga ctaagtgaag tgttctacac aggcaggaaa gacacctcaa ggctgtgcct
     3361 ttgtgcttag ttccatagag ttgaagactc agagaactaa ttttattttc taatttgtgg
     3421 atcaatagat tcagatcaat taaaccagag catcagggaa tgataggatg ctgtggacta
     3481 aatgtccgtt tttctccctg ttttcattct ccatgacacc ttcccctgtt gcacaggatc
     3541 tggcaagtct gaaaatccct gagcaatttc gacatgcgat ctggaagggc atcctggacc
     3601 accggcagct ccacgaattc tcctcccctt ctcatctcct gcggacccca agcagtgcct
     3661 ctacagtcag tgtgggctcc agtgagaccc ggggtgagcg tgttattgat gctgtgcgat
     3721 tcaccctccg ccagaccatc tctttcccac cccgagatga gtggaatgac ttcaactttg
     3781 acatggatgc tcgccgcaat aagcaacagc gcatcaaaga ggagggggag gggattccaa
     3841 ctactgcaag cgagaatctt tattttcagg gcgccgccaa attcaaagaa accgctgctg
     3901 ctaaattcga acgccagcac atggacagcg gaggtggagg ttcgagcggt ccctcgggtt
     3961 cgtcgagcct ggaagttctg ttccaggggc ccctgtcgtc gagcggtccc tcgggttcga
     4021 tggtgagcaa aggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     4081 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     4141 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     4201 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     4261 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     4321 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     4381 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     4441 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     4501 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     4561 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     4621 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     4681 ttaaggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaat
     4741 gatctagagt cgagttaatt aagaattccg cccccccccc tctccctccc cccccctaac
     4801 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc
     4861 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg
     4921 agcattccta ggggtctttc ccctctcgcc aaaggaatgc agggtctgtt gaatgtcgtg
     4981 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc
     5041 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa
     5101 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa
     5161 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta
     5221 ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg
     5281 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca
     5341 cgatgataat atggccacaa cctgttacat tgcacaagat aaaaatatat catcacgaac
     5401 agtaaaactg tctgcttaca taaacagtaa tacaaggggt gttatgggat cggccattga
     5461 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     5521 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     5581 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     5641 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     5701 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     5761 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     5821 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     5881 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     5941 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     6001 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     6061 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     6121 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     6181 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     6241 cttctgagtc gcctcaccat gtgagctctt cctatccctc tcctaactgc cagcccccta
     6301 aaagcactcc tgcttaatct tcaaagcctt ctccctagct cctccccttc ctcttgtctg
     6361 atttcttagg ggaaggagaa gtaagaggct acctcttacc taacatctga cctggcatct
     6421 aattctgatt ctggctttaa gccttcaaaa ctatagcttg cagaactgta gctgccatgg
     6481 ctaggtagaa gtgagcaaaa aagagttggg tgtctcctta agctgcagag atttctcatt
     6541 gacttttata aagcatgttc acccttatag tctaagacta tatatataaa tgtataaata
     6601 tacagtatag atttttgggt ggggggcatt gagtattgtt taaaatgtaa tttaaatgaa
     6661 agaaaattga gttgcactta ttgaccattt tttaatttac ttgttttgga tggcttgtct
     6721 atactccttc ccttaagggg tatcatgtat ggtgataggt atctagagct taatgctaca
     6781 tgtgagtgac gatgatgtac agattctttc agttctttgg attctaaata catgccacat
     6841 caaacctttg agtagatcca tttccattgc ttattatgta ggtaagactg tagatatgta
     6901 ttcttttctc agtgttggta tattttatat tactgacatt tcttctagtg atgatggttc
     6961 acgttggggt gatttaatcc agttataaga agaagttcat gtccaaacgt cctctttagt
     7021 ttttggttgg gaatgaggaa aattcttaaa aggcccatag cagccagttc aaaaacaccc
     7081 gacgtcatgt atttgagcat atcagtaacc cccttaaatt taataccaga taccttatct
     7141 tacaatattg attgggaaaa catttgctgc cattacagag gtattaaaac taaatttcac
     7201 tactagattg actaactcaa atacacattt gctactgttg taagaattct gattgatttg
     7261 attgggatga atgccatcta tctagttcta acagtgaagt tttactgtct attaatattc
     7321 agggtaaata ggaatcattc a
//
