LOCUS       sequence-341784-        4448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector for Gateway cloning that contains an mCherry 
            fused with an optimized photoactivatable Rac1 gene insert.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4448)
  AUTHORS   Harris JM, Wang AY, Boulanger-Weill J, Santoriello C, Foianini S, 
            Lichtman JW, Zon LI, Arlotta P
  TITLE     Long-Range Optogenetic Control of Axon Guidance Overcomes 
            Developmental Boundaries and Defects.
  JOURNAL   Dev Cell. 2020 Jun 8;53(5):577-588.e7. doi: 
            10.1016/j.devcel.2020.05.009.
  PUBMED    32516597
REFERENCE   2  (bases 1 to 4448)
  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"; doi: 
            "10.1016/j.devcel.2020.05"; journalName: "Dev Cell"; date: 
            "2020-06-8- 8"; volume: "53"; issue: "5"; pages: "577-588.e7"
COMMENT     Sequence Label: pME-mCherry-PA-Rac1
FEATURES             Location/Qualifiers
     source          1..4448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      837..1425
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1326..1345
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1579..1596
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(1755..1782)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1874..1960)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2009..2031
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2023..2040
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2024..2040
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2056..2155
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2310..2327
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2334..3038
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2476..2494)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2744..2763
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3045..3473
                     /codon_start=1
                     /gene="NPH1 from Avena sativa"
                     /product="light, oxygen, or voltage domain from phototropin
                     1 of Avena sativa (Herrou and Crosson, 2011)"
                     /label=LOV2
                     /note="photoswitch that undergoes a light-inducible 
                     conformational change"
                     /translation="LATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQ
                     YFIGVQKDGTEHVRDAAEREGVMEIKKTAENIDEAAKEL"
     protein_bind    complement(4060..4159)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4176..4195)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4177..4195)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4200..4216)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4200..4216)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4329..4448,1..690)
                     /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(4396..4415)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
       61 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
      121 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
      181 ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
      241 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
      301 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
      361 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
      421 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
      481 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
      541 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
      601 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
      661 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg
      721 cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg
      781 agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
      841 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
      901 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
      961 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     1021 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1081 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1141 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1201 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1261 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1321 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     1381 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     1441 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     1501 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     1561 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     1621 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     1681 gactggaaag cgggcagtga gcgcaacgca attaatacgc gtaccgctag ccaggaagag
     1741 tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt tgatgcctgg
     1801 cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca acgttcaaat
     1861 ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
     1921 gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca gttccctact
     1981 ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac gttgtaaaac gacggccagt
     2041 cttaagctcg ggccccaaat aatgatttta ttttgactga tagtgacctg ttcgttgcaa
     2101 caaattgatg agcaatgctt ttttataatg ccaactttgt acaaaaaagc aggctccgcg
     2161 gccaatctgc tcagagagga cagagtgggc aggagccagc attgggtata taaagctgag
     2221 cagggtcagt tgcttcttac gtttgcttct gagtctgttg tgttgacttg caacctcaga
     2281 aacagacatc cgcccccttc accatggcac accatcacca ccatcacgga tctgtgagca
     2341 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     2401 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     2461 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     2521 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     2581 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     2641 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     2701 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     2761 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     2821 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     2881 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     2941 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     3001 gccgccactc caccggcggc atggacgagc tgtacaaggg atccttggct actacacttg
     3061 aacgtattga gaagaacttt gtcattactg acccaagatt gccagataat cccattatat
     3121 tcgcgtccga tagtttcttg cagttgacag aatatagccg tgaagaaatt ttgggaagaa
     3181 actgcaggtt tctacaaggt cctgaaactg atcgcgcgac agtgagaaaa attagagatg
     3241 ccatagataa ccaaacagag gtcactgttc agctgattaa ttatacaaag agtggtaaaa
     3301 agttctggaa cctctttcac ttgcagccta tgcgagatca gaagggagat gtccagtact
     3361 ttattggggt tcagaaggat ggaactgagc atgtccgaga tgctgccgag agagagggag
     3421 tcatggagat taagaaaact gcagaaaata ttgatgaggc ggcaaaagaa cttatcaagt
     3481 gtgtggtggt gggagacgga gctgtaggta aaacttgcct actgatcagt tacacaacca
     3541 atgcatttcc tggagaatat atccctactg tctttgacaa ttattctgcc aatgttatgg
     3601 tagatggaaa accggtgaat ctgggcttat gggatacagc tggactagaa gattatgaca
     3661 gattacgccc cctatcctat ccgcaaacag atgtgttctt aatttgcttt tcccttgtga
     3721 gtcctgcatc attccaccac gtccgtgcaa agtggtatcc tgaggtgcgg caccactgtc
     3781 ccaacactcc catcatccta gtgggaacta aacttgatct tagggatgat aaagacacga
     3841 tcgagaaact gaaggagaag aagctgactc ccatcaccta tccgcagggt ctagccatgg
     3901 ctaaggagat tggtgctgta aaatacctgg agtgctcggc gctcacacag cgaggcctca
     3961 agacagtgtt tgacgaagcg atccgagcag tcctctgccc gcctcccgtg aagaagagga
     4021 agagaaaatg cctgctgttg taaaagggtg ggcgcgccga cccagctttc ttgtacaaag
     4081 ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc
     4141 aaaataaaat cattatttgc catccagctg atatccccta tagtgagtcg tattacatgg
     4201 tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac
     4261 aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacca
     4321 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg
     4381 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     4441 tctatcgc
//
