LOCUS       sequence-314982-        4786 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector for cloning Cryptochrome2-mCherry using 2-fragment 
            gateway recombination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4786)
  AUTHORS   Kaur P, Kibat C, Teo E, Gruber J, Mathuru A, Tolwinski ANS
  TITLE     Use of Optogenetic Amyloid-beta to Monitor Protein Aggregation in 
            Drosophila melanogaster, Danio rerio and Caenorhabditis elegans.
  JOURNAL   Bio Protoc. 2020 Dec 5;10(23):e3856. doi: 10.21769/BioProtoc.3856. 
            eCollection 2020 Dec 5.
  PUBMED    33659494
REFERENCE   2  (bases 1 to 4786)
  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: "Bio 
            Protoc."; date: "2020-12-5"; volume: "10(23):e3856. doi"; pages: " 
            10.21769/BioProtoc.3856. eCollection 2020 Dec 5"
COMMENT     Sequence Label: pDONR-attL5-CRY2-mCh-attL2
FEATURES             Location/Qualifiers
     source          1..4786
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    34..129
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             132..1625
                     /codon_start=1
                     /product="N-terminal photolyase homology region of 
                     Arabidopsis thaliana cryptochrome 2"
                     /label=CRY2PHR
                     /note="binds to CIB1 upon exposure to blue light (Kennedy 
                     et al., 2010)"
                     /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ
                     FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY
                     DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI
                     ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN
                     EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE
                     ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL
                     VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY
                     ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA
                     SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAA"
     regulatory      1644..1653
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1650..2360
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1795..1813)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2063..2082
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2362..2461)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2491..2510)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2492..2510)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2515..2531)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2515..2531)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2633..2652)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2644..3453
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2711..2730)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3600..4188
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4089..4108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4342..4359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4518..4545)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4637..4723)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(4772..4786,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4786,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag
       61 tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtata
      121 caaaagttgt aatgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga
      181 ttgaggataa tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca
      241 tttggtgtcc tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga
      301 aacaatcact tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa
      361 tcaaaaccca caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa
      421 aagtcgtctt taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg
      481 agaagctggt ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac
      541 cgtgggagat atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga
      601 aatgcttaga tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa
      661 taactgcagc ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg
      721 aggccgagaa accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg
      781 ctgataagtt actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca
      841 agaaagttgt tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa
      901 gcgtcagaca cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga
      961 acagtgaagg agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt
     1021 ctcggtatat atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc
     1081 ggtttttccc ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg
     1141 gttatccgtt ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca
     1201 gaataagagt gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg
     1261 gaatgaagta tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct
     1321 ggcagtatat ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg
     1381 cgttacaagg cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc
     1441 ttgcgagatt gccaactgaa tggatccatc atccatggga cgctccttta accgtactca
     1501 aagcttctgg tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag
     1561 ctcgtgagct actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag
     1621 cagcagcccg ggatccaccg gtcgccacca tggtgagcaa gggcgaggag gataacatgg
     1681 ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg
     1741 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     1801 tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc cctcagttca
     1861 tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     1921 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     1981 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     2041 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     2101 cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag cagaggctga
     2161 agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag gccaagaagc
     2221 ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc tcccacaacg
     2281 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     2341 tggacgagct gtacaagtag aacccagctt tcttgtacaa agttggcatt ataagaaagc
     2401 attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt
     2461 gccatccagc tgcagggcgg ccgcgatatc ccctatagtg agtcgtatta catggtcata
     2521 gctgtttcct ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat
     2581 aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     2641 gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct
     2701 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     2761 cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     2821 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     2881 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     2941 cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     3001 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     3061 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     3121 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     3181 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     3241 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     3301 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     3361 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     3421 cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac
     3481 tggcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa atcccttaac
     3541 gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3601 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3661 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3721 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3781 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3841 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3901 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3961 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4021 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4081 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4141 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4201 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4261 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4321 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     4381 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     4441 cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc tagccaggaa
     4501 gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc
     4561 tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca
     4621 aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa
     4681 aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct
     4741 actctcgcgt taacgctagc atggatgttt tcccagtcac gacgtt
//
