LOCUS       sequence-347860-        7217 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 7217)
  TITLE     ENCODE collection: CRISPR constructs
REFERENCE   2  (bases 1 to 7217)
  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: pSOX11-donor
FEATURES             Location/Qualifiers
     source          1..7217
                     /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             3821..3841
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3854..3898
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3938..3961
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(3989..3991,3992..3994,3995..4708)
                     /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(4034..4055)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4295..4314)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(4746..5319,5320..5322,5323..5331)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4912..4929)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5139..5158
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5413..6216
                     /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(5476..5495)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6086..6105
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(6286..6303)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(6307..6327)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
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 gaagatcatg gagcagtctc cggacatgca
     2701 caacgccgag atctccaaga ggctgggcaa gcgctggaaa atgctgaagg acagcgagaa
     2761 gatcccgttc atccgggagg cggagcggct gcggctcaag cacatggccg actaccccga
     2821 ctacaagtac cggccccgga aaaagcccaa aatggacccc tcggccaagc ccagcgccag
     2881 ccagagccca gagaagagcg cggccggcgg cggcggcggg agcgcgggcg gaggcgcggg
     2941 cggtgccaag acctccaagg gctccagcaa gaaatgcggc aagctcaagg cccccgcggc
     3001 cgcgggcgcc aaggcgggcg cgggcaaggc ggcccagtcc ggggactacg ggggcgcggg
     3061 cgacgactac gtgctgggca gcctgcgcgt gagcggctcg ggcggcggcg gcgcgggcaa
     3121 gacggtcaag tgcgtgtttc tggatgagga cgacgacgac gacgacgacg acgacgagct
     3181 gcagctgcag atcaaacagg agccggacga ggaggacgag gaaccaccgc accagcagct
     3241 cctgcagccg ccggggcagc agccgtcgca gctgctgaga cgctacaacg tcgccaaagt
     3301 gcccgccagc cctacgctga gcagctcggc ggagtccccc gagggagcga gcctctacga
     3361 cgaggtgcgg gccggcgcga cctcgggcgc cgggggcggc agccgcctct actacagctt
     3421 caagaacatc accaagcagc acccgccgcc gctcgcgcag cccgcgctgt cgcccgcgtc
     3481 ctcgcgctcg gtgtccacct cctcgtccag cagcagcggc agcagcagcg gcagcagcgg
     3541 cgaggacgcc gacgacctga tgttcgacct gagcttgaat ttctctcaaa gcgcgcacag
     3601 cgccagcgag cagcagctgg ggggcggcgc ggcggccggg aacctgtccc tgtcgctggt
     3661 ggataaggat ttggattcgt tcagcgaggg cagcctgggc tcccacttcg agttccccga
     3721 ctactgcacg ccggagctga gcgagatgat cgcgggggac tggctggagg cgaacttctc
     3781 cgacctggtg ttcacatatg ggattccaac tactgcaagc gagaatcttt attttcaggg
     3841 cgccgccaaa ttcaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagcgg
     3901 aggtggaggt tcgagcggtc cctcgggttc gtcgagcctg gaagttctgt tccaggggcc
     3961 cctgtcgtcg agcggtccct cgggttcgat ggtgagcaaa ggcgaggagc tgttcaccgg
     4021 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     4081 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     4141 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     4201 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     4261 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     4321 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     4381 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     4441 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     4501 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     4561 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     4621 ccccaacgag aagcgcgatc acatggtcct taaggagttc gtgaccgccg ccgggatcac
     4681 tctcggcatg gacgagctgt acaagtaatg atctagagtc gagttaatta agaattccgc
     4741 ccccccccct ctccctcccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     4801 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     4861 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     4921 aaggaatgca gggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     4981 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     5041 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     5101 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     5161 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     5221 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     5281 acggggacgt ggttttcctt tgaaaaacac gatgataata tggccacaac ctgttacatt
     5341 gcacaagata aaaatatatc atcacgaaca gtaaaactgt ctgcttacat aaacagtaat
     5401 acaaggggtg ttatgggatc ggccattgaa caagatggat tgcacgcagg ttctccggcc
     5461 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     5521 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5581 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     5641 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5701 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5761 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5821 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5881 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5941 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     6001 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     6061 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     6121 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     6181 atcgccttct atcgccttct tgacgagttc ttctgagtca aggcgcccgc tgctcgctct
     6241 ttctctcgga gggtgcagag ctgggttcct tgggaggaag ttgtagtggt gatgatgatg
     6301 atgataatga tgatgatgat ggtggtgttg atggtggcgg tggtagggtg gaggggagag
     6361 aagaagatgc tgatgatatt gataagatgt cgtgacgcaa agaaattgga aaacatgatg
     6421 aaaattttgg tggagttaaa gtgaaatgag tagtttttaa acatttttcc tgtccttttt
     6481 ttgtcccccc tcccttcctt tatcgtgtct caaggtagtt gcatacctag tctggagttg
     6541 tgattatttt cccaaaaaat gtgtttttgt aattactatt tctttttcct gaaattcgtg
     6601 attgcaacaa aggcagaggg ggcggggcgg gggaggggag gtaggacccg ctccggaagg
     6661 cgctgtttga agcttgtcgg tctttgaagt ctggaagacg tctgcagagg acccttttgg
     6721 cagcacaact gttactctag ggagttggtg gagatatttt tttttcttaa gagaacttaa
     6781 agaactggtg attttttttt aacaaaaaaa gggaccattg caacttttgt taatttaatt
     6841 tttttttttt tttttttttt tttttttgga gggagaaaac tgatgtcttc tatgcatccg
     6901 attcttaaca aaactgcagg gagcttgaaa aaatgcagac tgtacaaacg cttacaaaaa
     6961 aaaaaactgt gaactgactt aagatcagag tttacttttc agatcaaatt gtttatggtt
     7021 ttacaaatgt gatttctact tgccaacttt ttttttgtaa cttgttccct tatacctcct
     7081 tgattgaata ccagacagcc tagacctcag tacaaaaggt attgaaacat ttttgataca
     7141 taacagacct cagtcttttt taaaaattaa tatattttca ggcgtatttt tgtacagtga
     7201 aaagggaaca ttcttgc
//
