LOCUS       sequence-483116-        5067 bp    DNA     circular SYN 19-AUG-2025
DEFINITION  Allows for mCherry::RBD::T2A::rac-2::mGFP entry into Gateway 
            Vectors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5067)
  AUTHORS   Lamb R, Scales M, Watkins J, Werner M, Cherra SJ
  TITLE     A PDZ-RapGEF promotes synaptic development in C. elegans through a 
            Rap/Rac signaling pathway.
  JOURNAL   Development. 2025 Aug 1:dev.204678. doi: 10.1242/dev.204678.
  PUBMED    40748210
REFERENCE   2  (bases 1 to 5067)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 19, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Development. 2025 Aug 1:dev.204678. doi: 10.1242/dev.204678."
COMMENT     Sequence Label: pREA12
FEATURES             Location/Qualifiers
     source          1..5067
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1489..1554
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(2197..2199,2200..2202,2203..2916)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2242..2263)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2503..2522)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2850..2871
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2946..3045)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3062..3081)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3063..3081)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3086..3102)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3086..3102)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3533..4324
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      4417..5005
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4906..4925
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc gaattcgccc ttggactagt catatgatgg tctcaaaggg tgaagaagat
      721 aacatggcaa ttattaaaga gtttatgcgt ttcaaggtgc atatggaggg atctgtcaat
      781 gggcatgagt ttgaaattga aggtgaagga gaaggccgac catatgaggg aacacaaacc
      841 gcaaaactaa aggtaactaa aggcggacca ttaccattcg cctgggacat cctctctcca
      901 cagttcatgt atggaagtaa agcttatgtt aaacatccgg cagatatacc agattatttg
      961 aaactttcat tcccggaggg ttttaagtgg gaacgcgtaa tgaattttga agacggagga
     1021 gttgttacag tgacgcaaga ctcaagcctc caagatggag aatttattta taaagtcaaa
     1081 cttcgaggaa cgaatttccc ctcggatgga cctgttatgc agaagaagac tatgggatgg
     1141 gaagcttcaa gtgaaagaat gtaccctgaa gacggtgctc ttaagggaga gattaaacaa
     1201 cgtcttaaat tgaaagatgg aggacattac gatgctgagg tgaagacaac ttacaaagcc
     1261 aaaaaaccag ttcagctgcc aggagcgtac aatgttaata ttaaactgga tatcacctcc
     1321 cacaacgagg attacactat cgttgagcaa tatgaaagag ctgaagggcg gcactcgaca
     1381 ggtggcatgg atgaattgta taagactagt ggttccggag gatctggagg atctggtacc
     1441 atctctcgac cgagcaattt cgaacacaca attcatgtcg gaggtaccgt aaaacaaacg
     1501 ctcaactttg acttacttaa acttgcgggc gacgttgagt caaacccagg accaggatcc
     1561 atgcaagcaa tcaaatgtgt cgtcgttggt gacggagccg tcggtaagac gtgtctcctg
     1621 ctatcgtaca ctacaaacgc ttttcccgga gaatatattc taacggtatt cgacacctac
     1681 tcaacaaatg tgatggtcga cggaaggcca ataaatctca gcctatggga cacagctgga
     1741 caggacgatt acgatcaatt ccgccacctg tcatttccac aaacagacgt attcctcgta
     1801 tgctttgcac tgaataatcc agcaagtttt gagaatgttc gtgcaaaatg gtatccagaa
     1861 gtatcacatc attgcccaaa tacaccgatt attttggttg gaactaaagc tgatttacgc
     1921 gaggatcgag atactattga acggctccgt gaacgtcggc tccaaccagt gagccacacc
     1981 cagggttacg tgatggcaaa ggaaatcaag gcggtcaagt acctggaatg ctcggcgctt
     2041 acccaaattg gattgaaaca agttttcgat gaggcaattc gtactgggct caccccgcca
     2101 caaacaccac aaacgagagc caaaaagagc aattgcacgg tgcttggtgg ttccggtgga
     2161 tccggttccg gaggatctgg aggatctgcg gccgcgatgg tgagcaaggg cgaggagctg
     2221 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2281 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     2341 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     2401 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2461 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2521 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2581 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     2641 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     2701 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     2761 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg
     2821 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2881 gggatcactc tcggcatgga cgagctgtac aagtagcata tggcggccgc ggaagggcga
     2941 attcgaccca gctttcttgt acaaagttgg cattataaaa aataattgct catcaatttg
     3001 ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgatat
     3061 cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc
     3121 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgcc tcctctagac
     3181 cagccaggac agaaatgcct cgacttcgct gctgcccaag gttgccgggt gacgcacacc
     3241 gtggaaacgg atgaaggcac gaacccagtg gacataagcc tgttcggttc gtaagctgta
     3301 atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg
     3361 taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgggg tacagtctat
     3421 gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag
     3481 cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca tcatgaggga
     3541 agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca
     3601 tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa
     3661 gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg
     3721 gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct
     3781 ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc
     3841 taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga
     3901 gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt
     3961 tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt
     4021 tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga
     4081 gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc
     4141 gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt
     4201 catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc
     4261 agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa
     4321 ataaccctcg agccacccat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     4381 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4441 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4501 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4561 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4621 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4681 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4741 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4801 gcgtgagcat tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4861 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4921 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4981 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5041 cttttgctgg ccttttgctc acatgtt
//
