LOCUS       sequence-347870-        7325 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 7325)
  TITLE     ENCODE collection: CRISPR constructs
REFERENCE   2  (bases 1 to 7325)
  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: pBACH2-donor
FEATURES             Location/Qualifiers
     source          1..7325
                     /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             3816..3836
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3849..3893
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             3933..3956
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(3984..3986,3987..3989,3990..4703)
                     /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(4029..4050)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4290..4309)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(4741..5314,5315..5317,5318..5326)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4907..4924)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5134..5153
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5408..6211
                     /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(5471..5490)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6081..6100
                     /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 cccaagtcca tagtttgcat tagagttcac
     2701 tctcggtgtt gcacgttcta tggttttgga caaatgtata atgacatgga tccaccagta
     2761 tagtgtcata cagagtggct tcactgcccc caaatcctct gtgctccacc tgttcaaccc
     2821 cttttacccc ttgcaaccac agatcttttt actgcttcca tagttttgct tttttcgaaa
     2881 tgtcatacgg ttggaatggt agatttttgc ctttcaaaaa tggcagcctg atacatttgg
     2941 gggagatttt cagtcacaag attgggactg agagtttttc atttaactct ctacaaaccc
     3001 caaattccag gaaaccaaaa cattgactct gaagttttct gattcgtgac gattttgccg
     3061 gttgtttgcc cgggccttgg agagggatga agagatgtcc tggggaaggt gttggcatca
     3121 gatggaaggt gtggcagaag cctgtgtctg ggccccctga atatgtatct ttaagtttct
     3181 agagcctgac ctagtcattc ttgagttaca ttataatgga ggacgcaaaa caaaggctgg
     3241 aaaacctgaa acgaggcctt ctgtcctcca gttcaggctt gatcaaccct tctctttttt
     3301 tctctactgt ccaggtgtgt gagaaagaga aactgttgtc agagaggaat caactgaaag
     3361 catgcatggg ggaactgttg gacaacttct cctgcctttc ccagggagtt tgccgagaca
     3421 tccagagccc cgagcagatc caggccctgc atcggtattg ccctgtcctc agacccatgg
     3481 acttgcccac ggcctccagt attaaccctg cgcccttggg tgctgagcag aacattgcgg
     3541 cctcccaatg cgcagtgggg gaaaacgtgc cctgctgctt ggagccaggc gcggctcccc
     3601 ccggaccccc ctgggcaccc agcaacacct ccgagaattg tacctctggg aggagactag
     3661 aaggcactga cccgggaacc ttctcagaga gaggacctcc tcttgaaccc aggagccaaa
     3721 cagtgaccgt ggacttctgc caggaagtga ctgataagtg tacaactgac gaacagccca
     3781 ggaaagatta taccgggatt ccaactactg caagcgagaa tctttatttt cagggcgccg
     3841 ccaaattcaa agaaaccgct gctgctaaat tcgaacgcca gcacatggac agcggaggtg
     3901 gaggttcgag cggtccctcg ggttcgtcga gcctggaagt tctgttccag gggcccctgt
     3961 cgtcgagcgg tccctcgggt tcgatggtga gcaaaggcga ggagctgttc accggggtgg
     4021 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     4081 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     4141 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     4201 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     4261 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     4321 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     4381 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     4441 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     4501 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     4561 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     4621 acgagaagcg cgatcacatg gtccttaagg agttcgtgac cgccgccggg atcactctcg
     4681 gcatggacga gctgtacaag taatgatcta gagtcgagtt aattaagaat tccgcccccc
     4741 cccctctccc tccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
     4801 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     4861 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     4921 atgcagggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
     4981 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     5041 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     5101 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     5161 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     5221 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
     5281 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaacctgtt acattgcaca
     5341 agataaaaat atatcatcac gaacagtaaa actgtctgct tacataaaca gtaatacaag
     5401 gggtgttatg ggatcggcca ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     5461 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     5521 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     5581 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     5641 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     5701 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     5761 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     5821 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     5881 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     5941 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     6001 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     6061 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     6121 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     6181 cttctatcgc cttcttgacg agttcttctg agtctgactc ggctctgcct cccagtccgc
     6241 acacctctcc catccaggcg ttcttcagtc agcctgtggc actgttcatc tgctgtcccg
     6301 aagaaaccga gaacacattt ggtgcacact acagcggtct tagcagcaat actgttccga
     6361 agtatcctct cctcttctcg agcaggagtg atagttacct tcacaatggt gctacccctt
     6421 gcccaggcaa ggaaagacag cagtgatgac actgtctgtc tgtggctcaa tttcagtctt
     6481 cacagggata gactacaaca cccctaggcc ccaaccacgg attttttttc tcagtggccc
     6541 atgtcacaaa ccctatctca ggaatttctt ctgaatgttc aatttttttc attgaagaca
     6601 gcttctatac acatcaaagt tttatagcta gactgtacat attatatata atatatatat
     6661 aaaatatata tatatatata tatatccata tgcaaaagtc ctgcatgcct caactttctc
     6721 atcctaaaac tggaaactta tttctcattt agaaacaggt tccaacattc ctcttctttt
     6781 gtctctgatg ctagaactag tttggtaact gttaacatgg tcatttttct tgcttcacag
     6841 ttcaattttc aattcgtact tatttatgga caaaattcag tgttggaagc tttttcccaa
     6901 ggttttattt cagatttctt tttcgtttgg tttggttttg gcacctccaa gtggtgtcat
     6961 ttgagcattg taggtttgtt ttttgtttgt ttggggggtt ttgtttgttt ttgtttttgt
     7021 ttttgttttc cttgcagata ctgtacagta atggtcaact ttgccacttg cactgagttt
     7081 tgggtcaaac ctattttctc aaatgaagtt gtaacttcgg tataactcaa gtatactgta
     7141 tattctttgc ttttagttaa aaaagtaaaa cattttagct aattaaaaag cactcaggtg
     7201 ataattatgt aggaaaaaca atcttaccaa ataatgaatt catcctagga tgtgtagaca
     7261 ataatctgct tgaatgtttt tatatttcac ctcctcccca cctttcccta agaaagttta
     7321 aacgc
//
