LOCUS       sequence-347400-        7252 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 7252)
  TITLE     ENCODE collection: CRISPR constructs
REFERENCE   2  (bases 1 to 7252)
  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: pPITX2-donor
FEATURES             Location/Qualifiers
     source          1..7252
                     /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             3822..3842
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3855..3899
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3939..3962
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(3990..3992,3993..3995,3996..4709)
                     /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(4035..4056)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4296..4315)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(4747..5320,5321..5323,5324..5332)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4913..4930)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5140..5159
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5414..6217
                     /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(5477..5496)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6087..6106
                     /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 tcggtaccct gctaggcgag aaagagtgtt ggcggcctag
     2701 tccctgttaa gggagcacgt accagggggt gggggacgac agtggaggtc agggaaggaa
     2761 gggaggaatt gcgtgggaga aagagcgatc ctctagtgcc cttccagccc cttctcctca
     2821 tccgtgggtc tgtggctttg gaatggaagc aagtttgcaa ggtgccccgg gaagggttgg
     2881 aaaagcctgc tgccccgcgt ttgttttaca ttaagtgttt ttggacctgg agaaacgcct
     2941 ggctgggtga tcaaaccgtc cgcaggtctc catgcgttcg gctgaggttt gtggcgtagc
     3001 tccgagtccc agctcgcagg ccagagcaga ccaggtctcc tgcgcttggt ggagacccgg
     3061 gccagtaact gaaagctggc cctggtatct tggtgtgcag ggcggtgcag tgaagcgagg
     3121 ccagggtgtg tgagtgcgct agcgtgtgtg tcgggggaag gcgggggttg gcctccgatg
     3181 gaagttttag taatctgcac tgtggcatct gtttgctccc ttgccccaac cgcccccagg
     3241 tttggttcaa gaatcgtcgg gccaaatgga gaaagaggga gcgcaaccag caggccgagc
     3301 tacgcaagaa tggcttcggg ccgcagttca atgggctcat gcagccctac gacgacatgt
     3361 acccaggcta ttcctacaac aactgggccg ccaagggcct tacatccgcc tccctatcca
     3421 ccaagagctt ccccttcttc aactctatga acgtcaaccc cctgtcatca cagagcatgt
     3481 tttccccacc caactctatc tcgtccatga gcatgtcgtc cagcatggtg ccctcagcag
     3541 tgacaggcgt cccgggctcc agtctcaaca gcctgaataa cttgaacaac ctgagtagcc
     3601 cgtcgctgaa ttccgcggtg ccgacgcctg cctgtcctta cgcgccgccg actcctccgt
     3661 atgtttatag ggacacgtgt aacccgagcc tggccagcct gagactgaaa gcaaagcagc
     3721 actccagctt cggctacgcc agcgtgcaga acccggcctc caacctgagt gcttgccagt
     3781 atgcagtgga ccggcccgtg gggattccaa ctactgcaag cgagaatctt tattttcagg
     3841 gcgccgccaa attcaaagaa accgctgctg ctaaattcga acgccagcac atggacagcg
     3901 gaggtggagg ttcgagcggt ccctcgggtt cgtcgagcct ggaagttctg ttccaggggc
     3961 ccctgtcgtc gagcggtccc tcgggttcga tggtgagcaa aggcgaggag ctgttcaccg
     4021 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     4081 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     4141 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     4201 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     4261 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     4321 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     4381 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     4441 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     4501 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     4561 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     4621 accccaacga gaagcgcgat cacatggtcc ttaaggagtt cgtgaccgcc gccgggatca
     4681 ctctcggcat ggacgagctg tacaagtaat gatctagagt cgagttaatt aagaattccg
     4741 cccccccccc tctccctccc cccccctaac gttactggcc gaagccgctt ggaataaggc
     4801 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg
     4861 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc
     4921 aaaggaatgc agggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga
     4981 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg
     5041 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag
     5101 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc
     5161 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct
     5221 cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac
     5281 cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa cctgttacat
     5341 tgcacaagat aaaaatatat catcacgaac agtaaaactg tctgcttaca taaacagtaa
     5401 tacaaggggt gttatgggat cggccattga acaagatgga ttgcacgcag gttctccggc
     5461 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     5521 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     5581 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     5641 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     5701 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     5761 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     5821 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5881 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5941 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     6001 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     6061 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     6121 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     6181 catcgccttc tatcgccttc ttgacgagtt cttctgagtc gacgcaccca cagcgccggg
     6241 atcctaggac cttgccggat ggggcaactc cgcccttgaa agactgggaa ttatgctaga
     6301 aggtcgtggg cactaaagaa agggagagaa agagaagcta tatagagaaa aggaaaccac
     6361 tgaatcaaag agagagctcc tttgatttca aagggatgcc ctcagtgtct gacatctttc
     6421 actacaagta tttctaacag ttgcaaggac acatacacaa acaaatgttt gactggatat
     6481 gacattttaa cattactata agcttgttat tttttaagtt tagcattgtt aacatttaaa
     6541 tgactgaaag gatgtatata tatcgaaatg tcaaattaat tttataaaag cagttgttag
     6601 taatatcaca acagtgtttt taaaggttag gctttaaaat aaagcatgtt atacagaagc
     6661 gattaggatt tttcgcttgc gagcaaggga gtgtatatac taaatgccac actgtatgtt
     6721 tctaacatat tattattatt ataaaaaatg tgtgaatatc agttttagaa tagtttctct
     6781 ggtggatgca atgatgtttc tgaaactgct atgtacaacc taccctgtgt ataacatttc
     6841 gtacaatatt attgttttac ttttcagcaa atatgaaaca aatgtgtttt atttcatggg
     6901 agtaaaatat actgcataca aattggtctg gattctttct cccctcctct gtcactaact
     6961 tggccaggac atctcagtca ctgcttccta aacaaaccag ttccctctgc ctgcctagtt
     7021 aaacatacaa ggcagcagtc cttatttaaa tttggtagaa ataaatgata gccattcatc
     7081 agaaactaaa aagaaaaaaa aaaggcattc ccggggggga aaagggctac aaaatctaat
     7141 tttgtctctc caatttttct ttggcttaaa cctagaggat tccattatgg ctagcaaata
     7201 atgtgaaaaa gaaaaaagaa gaaagaaatt tagcaagtcc atcagcttaa aa
//
