LOCUS       sequence-97053-e        7541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cry2FL-VP64 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7541)
  AUTHORS   Polstein LR, Gersbach CA
  TITLE     A light-inducible CRISPR-Cas9 system for control of endogenous gene 
            activation.
  JOURNAL   Nat Chem Biol. 2015 Feb 9. doi: 10.1038/nchembio.1753.
  PUBMED    25664691
REFERENCE   2  (bases 1 to 7541)
  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"; journalName: "Nat Chem 
            Biol. 2015 Feb 9. doi: 10.1038/nchembio.1753."
COMMENT     Sequence Label: pcDNA3.1-Cry2FL-VP64
FEATURES             Location/Qualifiers
     source          1..7541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             907..972
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             979..999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1006..2841
                     /codon_start=1
                     /product="Arabidopsis thaliana cryptochrome 2"
                     /label=CRY2
                     /note="binds to CIB1 upon exposure to blue light (Kennedy 
                     et al., 2010)"
                     /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ
                     FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY
                     DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI
                     ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN
                     EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE
                     ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL
                     VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY
                     ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA
                     SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKE
                     PGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSV
                     FFVSQSCSLASEGKNLEGIQDSSDQITTSLGKNGCK"
     CDS             2866..2886
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2896..3045
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             3052..3078
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3135..3152)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3141..3365
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3411..3839
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3498..3517)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3708..3729
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3848..3868)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3853..4182
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4033..4168
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4095..4114
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4249..5043
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4303..4322)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4913..4932
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5217..5338
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5254..5273)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5308..5327
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5387..5403)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5387..5403)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5400..5422)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5411..5427
                     /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(5435..5441,5442..5459,5460..5465))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5480..5501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5618..5635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5789..6374)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5869..5888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6545..7336,7337..7405))
                     /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     7168..7187
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7406..7510)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
      961 gacgatgaca agatggcccc caagaagaag aggaaggtgg gccgcatgaa gatggacaaa
     1021 aagactatag tttggtttag aagagaccta aggattgagg ataatcctgc attagcagca
     1081 gctgctcacg aaggatctgt ttttcctgtc ttcatttggt gtcctgaaga agaaggacag
     1141 ttttatcctg gaagagcttc aagatggtgg atgaaacaat cacttgctca cttatctcaa
     1201 tccttgaagg ctcttggatc tgacctcact ttaatcaaaa cccacaacac gatttcagcg
     1261 atcttggatt gtatccgcgt taccggtgct acaaaagtcg tctttaacca cctctatgat
     1321 cctgtttcgt tagttcggga ccataccgta aaggagaagc tggtggaacg tgggatctct
     1381 gtgcaaagct acaatggaga tctattgtat gaaccgtggg agatatactg cgaaaagggc
     1441 aaacctttta cgagtttcaa ttcttactgg aagaaatgct tagatatgtc gattgaatcc
     1501 gttatgcttc ctcctccttg gcggttgatg ccaataactg cagcggctga agcgatttgg
     1561 gcgtgttcga ttgaagaact agggctggag aatgaggccg agaaaccgag caatgcgttg
     1621 ttaactagag cttggtctcc aggatggagc aatgctgata agttactaaa tgagttcatc
     1681 gagaagcagt tgatagatta tgcaaagaac agcaagaaag ttgttgggaa ttctacttca
     1741 ctactttctc cgtatctcca tttcggggaa ataagcgtca gacacgtttt ccagtgtgcc
     1801 cggatgaaac aaattatatg ggcaagagat aagaacagtg aaggagaaga aagtgcagat
     1861 ctttttctta ggggaatcgg tttaagagag tattctcggt atatatgttt caacttcccg
     1921 tttactcacg agcaatcgtt gttgagtcat cttcggtttt tcccttggga tgctgatgtt
     1981 gataagttca aggcctggag acaaggcagg accggttatc cgttggtgga tgccggaatg
     2041 agagagcttt gggctaccgg atggatgcat aacagaataa gagtgattgt ttcaagcttt
     2101 gctgtgaagt ttcttctcct tccatggaaa tggggaatga agtatttctg ggatacactt
     2161 ttggatgctg atttggaatg tgacatcctt ggctggcagt atatctctgg gagtatcccc
     2221 gatggccacg agcttgatcg cttggacaat cccgcgttac aaggcgccaa atatgaccca
     2281 gaaggtgagt acataaggca atggcttccc gagcttgcga gattgccaac tgaatggatc
     2341 catcatccat gggacgctcc tttaaccgta ctcaaagctt ctggtgtgga actcggaaca
     2401 aactatgcga aacccattgt agacatcgac acagctcgtg agctactagc taaagctatt
     2461 tcaagaaccc gtgaagcaca gatcatgatc ggagcagcac ctgatgagat tgtagcagat
     2521 agcttcgagg ccttaggggc taataccatt aaagaacctg gtctttgccc atctgtgtct
     2581 tctaatgacc aacaagtacc ttcggctgtt cgttacaacg ggtcaaagag agtgaaacct
     2641 gaggaagaag aagagagaga catgaagaaa tctaggggat tcgatgaaag ggagttgttt
     2701 tcgactgctg aatcttcttc ttcttcgagt gtgtttttcg tttcgcagtc ttgctcgttg
     2761 gcatcagaag ggaagaatct ggaaggtatt caagattcat ctgatcagat tactacaagt
     2821 ttgggaaaaa atggttgcaa accaattgcc ggatccaagg ctagcccgaa aaagaaacgc
     2881 aaagttgggc gcgccgacgc gctggacgat ttcgatctcg acatgctggg ttctgatgcc
     2941 ctcgatgact ttgacctgga tatgttggga agcgacgcat tggatgactt tgatctggac
     3001 atgctcggct ccgatgctct ggacgatttc gatctcgata tgttaattaa ctacccgtac
     3061 gacgttccgg actacgcttc ttgaatcggt aggaattcgc ggccgtctag acttaagttt
     3121 aaaccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3181 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3241 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3301 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3361 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3421 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3481 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3541 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3601 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3661 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3721 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3781 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     3841 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     3901 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3961 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4021 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4081 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     4141 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     4201 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     4261 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4321 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4381 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4441 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4501 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     4561 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     4621 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     4681 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     4741 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     4801 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     4861 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     4921 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     4981 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     5041 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     5101 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     5161 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     5221 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5281 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5341 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5401 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5461 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5521 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5581 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5641 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5701 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5761 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5821 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5881 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5941 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6001 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6061 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6121 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6181 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6241 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6301 gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6361 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6421 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6481 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6541 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6601 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6661 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6721 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6781 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6841 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6901 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6961 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7021 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7081 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7141 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7201 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7261 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7321 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7381 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7441 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7501 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
