LOCUS       sequence-215994-        7683 bp    DNA     circular SYN 14-APR-2025
DEFINITION  anchor iLID domain to plasma membrane with slower diffusion .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7683)
  AUTHORS   Van Geel O, Hartsuiker R, Gadella TWJ
  TITLE     Increasing spatial resolution of photoregulated GTPases through 
            immobilized peripheral membrane proteins.
  JOURNAL   Small GTPases. 2018 Sep 5:1-10. doi: 10.1080/21541248.2018.1507411.
  PUBMED    30182785
REFERENCE   2  (bases 1 to 7683)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Small 
            GTPases. 2018 Sep 5:1-10. doi: 10.1080/21541248.2018.1507411."
COMMENT     Sequence Label: CRY2clust-eGFP-2xC2-iLID
FEATURES             Location/Qualifiers
     source          1..7683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             597..2090
                     /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
                     SGVELGTNYAKPIVDIDTARELLAKAISRTRGAQIMIGAA"
     misc_feature    2118..2170
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      2178..2187
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2184..2186,2187..2189,2190..2900)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2184..2186,2187..2189,2190..2900)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2229..2250)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2490..2509)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2837..2858
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3900..4337
                     /codon_start=1
                     /product="improved light-inducible dimer when used with 
                     SspB nano or SspB micro (Guntas et al., 2015)"
                     /label=iLID
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNVFHLQPMRDYKGDVQ
                     YFIGVQLDGTERLHGAAEREAVCLIKKTAFQIAEAANDENYF"
     polyA_signal    4471..4592
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4508..4527)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4562..4581
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4599..5054)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4736..4757)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4948..4967
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5081..5185
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5187..5544
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5187..5378
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5210..5230)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5395..5530
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5579..6373
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5579..6373
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5633..5652)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6243..6262
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6561..6580)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6605..6652
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6981..7569
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7470..7489
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatga
      601 agatggacaa aaagactata gtttggttta gaagagacct aaggattgag gataatcctg
      661 cattagcagc agctgctcac gaaggatctg tttttcctgt cttcatttgg tgtcctgaag
      721 aagaaggaca gttttatcct ggaagagctt caagatggtg gatgaaacaa tcacttgctc
      781 acttatctca atccttgaag gctcttggat ctgacctcac tttaatcaaa acccacaaca
      841 cgatttcagc gatcttggat tgtatccgcg ttaccggtgc tacaaaagtc gtctttaacc
      901 acctctatga tcctgtttcg ttagttcggg accataccgt aaaggagaag ctggtggaac
      961 gtgggatctc tgtgcaaagc tacaatggag atctattgta tgaaccgtgg gagatatact
     1021 gcgaaaaggg caaacctttt acgagtttca attcttactg gaagaaatgc ttagatatgt
     1081 cgattgaatc cgttatgctt cctcctcctt ggcggttgat gccaataact gcagcggctg
     1141 aagcgatttg ggcgtgttcg attgaagaac tagggctgga gaatgaggcc gagaaaccga
     1201 gcaatgcgtt gttaactaga gcttggtctc caggatggag caatgctgat aagttactaa
     1261 atgagttcat cgagaagcag ttgatagatt atgcaaagaa cagcaagaaa gttgttggga
     1321 attctacttc actactttct ccgtatctcc atttcgggga aataagcgtc agacacgttt
     1381 tccagtgtgc ccggatgaaa caaattatat gggcaagaga taagaacagt gaaggagaag
     1441 aaagtgcaga tctttttctt aggggaatcg gtttaagaga gtattctcgg tatatatgtt
     1501 tcaacttccc gtttactcac gagcaatcgt tgttgagtca tcttcggttt ttcccttggg
     1561 atgctgatgt tgataagttc aaggcctgga gacaaggcag gacgggttat ccgttggtgg
     1621 atgccggaat gagagagctt tgggctaccg gatggatgca taacagaata agagtgattg
     1681 tttcaagctt tgctgtgaag tttcttctcc ttccatggaa atggggaatg aagtatttct
     1741 gggatacact tttggatgct gatttggaat gtgacatcct tggctggcag tatatctctg
     1801 ggagtatccc cgatggccac gagcttgatc gcttggacaa tcccgcgtta caaggcgcca
     1861 aatatgaccc agaaggtgag tacataaggc aatggcttcc cgagcttgcg agattgccaa
     1921 ctgaatggat ccatcatcca tgggacgctc ctttaaccgt actcaaagct tctggtgtgg
     1981 aactcggaac aaactatgcg aaacccattg tagacatcga cacagctcgt gagctactag
     2041 ctaaagctat ttcaagaacc cgtggagcac agatcatgat cggagcagca ccggatgaaa
     2101 ttgtggcgga tagctggctc gagctcaagc ttcgaattct gcagtcgacg gtaccgcggg
     2161 cccgggatcc accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg
     2221 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2281 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2341 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     2401 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2461 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2521 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2581 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2641 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2701 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2761 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     2821 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2881 gcatggacga gctgtacaag tccggaagta gttgcactga acccctaggc ctgaaggata
     2941 ataccatccc caacaagcag atcacagcct ccagctacta caaaacctgg ggcctgagtg
     3001 cctttagctg gtttccctac tacgcacgac tggataatca gggcaagttc aacgcctgga
     3061 ccgcccagac caacagtgcc tctgagtggc tgcagattga cctgggctcc cagaagcgag
     3121 tcacgggcat catcacccag ggtgcccgag actttggcca cattcaatat gtggctgcct
     3181 acagggtggc ctatggtgat gatggtgtga cctggactga gtacaaggac ccgggggcct
     3241 cagaaagcaa gattttccct ggtaacatgg acaataattc ccacaagaag aacatatttg
     3301 agacgccgtt ccaggctcgc ttcgtgcgga tccagcccgt ggcctggcac aaccgtatca
     3361 ccctgcgagt ggagctgctg ggctgtggta agcttagatc ttgcactgaa cccctaggcc
     3421 tgaaggataa taccatcccc aacaagcaga tcacagcctc cagctactac aaaacctggg
     3481 gcctgagtgc ctttagctgg tttccctact acgcacgact ggataatcag ggcaagttca
     3541 acgcctggac cgcccagacc aacagtgcct ctgagtggct gcagattgac ctgggctccc
     3601 agaagcgagt cacgggcatc atcacccagg gtgcccgaga ctttggccac attcaatatg
     3661 tggctgccta cagggtggcc tatggtgatg atggtgtgac ctggactgag tacaaggacc
     3721 cgggggcctc agaaagcaag attttccctg gtaacatgga caataattcc cacaagaaga
     3781 acatatttga gacgccgttc caggctcgct tcgtgcggat ccagcccgtg gcctggcaca
     3841 accgtatcac cctgcgagtg gagctgctgg gctgttatga attcagcatg ggggagtttc
     3901 tggcaaccac actggaacgg atcgagaaaa atttcgtgat tactgatccg agactgcctg
     3961 acaacccaat catttttgcg agcgattcct tcctgcagct gacagaatat tctcgggaag
     4021 agatcctggg gcgcaattgc cgttttctgc agggacccga gacagaccgt gccactgttc
     4081 ggaaaatcag agatgctatt gacaaccaga ctgaagtgac cgttcagctg atcaattata
     4141 ccaagagcgg caagaagttc tggaacgtgt tccacctgca gccgatgcgc gattataagg
     4201 gcgacgtcca gtacttcatt ggcgtgcagc tggatggcac cgaacgtctt catggcgccg
     4261 ctgagcgtga ggcggtctgc ctgatcaaaa agacagcctt tcagattgct gaggcagcga
     4321 acgacgaaaa ttactttggt acaagtaaag cggccgcgac tctagatcat aatcagccat
     4381 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     4441 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     4501 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4561 tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt
     4621 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     4681 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     4741 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     4801 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     4861 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     4921 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     4981 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     5041 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5101 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5161 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt
     5221 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     5281 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     5341 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     5401 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     5461 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     5521 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat
     5581 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     5641 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     5701 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     5761 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     5821 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     5881 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     5941 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     6001 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     6061 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     6121 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     6181 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     6241 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     6301 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     6361 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     6421 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     6481 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     6541 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     6601 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc
     6661 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa
     6721 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg
     6781 ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt
     6841 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6901 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6961 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     7021 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     7081 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     7141 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     7201 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     7261 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     7321 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7381 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7441 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7501 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7561 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7621 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg
     7681 cat
//
