LOCUS       sequence-486782-        6586 bp    DNA     circular SYN 19-SEP-2025
DEFINITION  Expresses mCherry::RBD::T2A::rac-2::mGFP in cholinergic motor 
            neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6586)
  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 6586)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 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: pREA16
FEATURES             Location/Qualifiers
     source          1..6586
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1293..1317
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2138..2203
                     /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(2846..2848,2849..2851,2852..3565)
                     /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(2891..2912)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3152..3171)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3499..3520
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3595..3619)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3645..3661)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3645..3661)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4398..4418)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4398..4416)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4437..4453)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4437..4453)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4450..4472)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4461..4477
                     /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(4485..4491,4492..4509,4510..4515))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4530..4551
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4668..4685)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4839..5427)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4919..4938)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5598..6389,6390..6458))
                     /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     6221..6240
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6459..6563)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgc
      661 ttttggtttt cacaattttc tggtttttgg gatttttgcg ggaattttga gaggagaaag
      721 ttgggagaga agatattgga atggtccccg gggagctgta catttataca ccaaggcaat
      781 cttcgtaaaa aataacaata gagtcatccc tcagaacaga aaagaaaccc gcccgggggc
      841 tcaaatacac atgtcatttt tcttttttcg cctgattttt agcctaaatt agtgtttagc
      901 aattttttaa ggtaactcgg tggagtcatt tgaacttttt aaactaaaat ttcagaaaaa
      961 attctttttt tttgtcaaaa aataattttc ccgccggaat ttttcaaaaa aataattttt
     1021 attttatttt tcaagttcct tgtttcctct ttattttttt attttgcccc caaaagtttt
     1081 acactaaaat atttaatttt caggtaattc atcgaaagtc tttctatttt ccgcatctct
     1141 tgttcaaaag cttgcatgcc tgcaggtcga ctctagagga tccccgggat tggccaaagg
     1201 acccaaaggt atgtttcgaa tgatactaac ataacataga acattttcag gaggaccctt
     1261 ggagggtacc ggtagaaaaa aagcttgata tcacaagttt gtacaaaaaa gcaggctccg
     1321 aattcgccct tggactagtc atatgatggt ctcaaagggt gaagaagata acatggcaat
     1381 tattaaagag tttatgcgtt tcaaggtgca tatggaggga tctgtcaatg ggcatgagtt
     1441 tgaaattgaa ggtgaaggag aaggccgacc atatgaggga acacaaaccg caaaactaaa
     1501 ggtaactaaa ggcggaccat taccattcgc ctgggacatc ctctctccac agttcatgta
     1561 tggaagtaaa gcttatgtta aacatccggc agatatacca gattatttga aactttcatt
     1621 cccggagggt tttaagtggg aacgcgtaat gaattttgaa gacggaggag ttgttacagt
     1681 gacgcaagac tcaagcctcc aagatggaga atttatttat aaagtcaaac ttcgaggaac
     1741 gaatttcccc tcggatggac ctgttatgca gaagaagact atgggatggg aagcttcaag
     1801 tgaaagaatg taccctgaag acggtgctct taagggagag attaaacaac gtcttaaatt
     1861 gaaagatgga ggacattacg atgctgaggt gaagacaact tacaaagcca aaaaaccagt
     1921 tcagctgcca ggagcgtaca atgttaatat taaactggat atcacctccc acaacgagga
     1981 ttacactatc gttgagcaat atgaaagagc tgaagggcgg cactcgacag gtggcatgga
     2041 tgaattgtat aagactagtg gttccggagg atctggagga tctggtacca tctctcgacc
     2101 gagcaatttc gaacacacaa ttcatgtcgg aggtaccgta aaacaaacgc tcaactttga
     2161 cttacttaaa cttgcgggcg acgttgagtc aaacccagga ccaggatcca tgcaagcaat
     2221 caaatgtgtc gtcgttggtg acggagccgt cggtaagacg tgtctcctgc tatcgtacac
     2281 tacaaacgct tttcccggag aatatattct aacggtattc gacacctact caacaaatgt
     2341 gatggtcgac ggaaggccaa taaatctcag cctatgggac acagctggac aggacgatta
     2401 cgatcaattc cgccacctgt catttccaca aacagacgta ttcctcgtat gctttgcact
     2461 gaataatcca gcaagttttg agaatgttcg tgcaaaatgg tatccagaag tatcacatca
     2521 ttgcccaaat acaccgatta ttttggttgg aactaaagct gatttacgcg aggatcgaga
     2581 tactattgaa cggctccgtg aacgtcggct ccaaccagtg agccacaccc agggttacgt
     2641 gatggcaaag gaaatcaagg cggtcaagta cctggaatgc tcggcgctta cccaaattgg
     2701 attgaaacaa gttttcgatg aggcaattcg tactgggctc accccgccac aaacaccaca
     2761 aacgagagcc aaaaagagca attgcacggt gcttggtggt tccggtggat ccggttccgg
     2821 aggatctgga ggatctgcgg ccgcgatggt gagcaagggc gaggagctgt tcaccggggt
     2881 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2941 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     3001 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     3061 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     3121 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     3181 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     3241 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     3301 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     3361 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3421 ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc
     3481 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3541 cggcatggac gagctgtaca agtagcatat ggcggccgcg gaagggcgca ttcgacccag
     3601 ctttcttgta caaagtggtg atatcgaatt cctgcagccc gggggatcca ctagttctag
     3661 agcggccgcc accgcggccg ctgtcatcag atcgccatct cgcgcccgtg cctctgactt
     3721 ctaagtccaa ttactcttca acatccctac atgctctttc tccctgtgct cccaccccct
     3781 atttttgtta ttatcaaaaa acttctctta atttctttgt tttttagctt cttttaagtc
     3841 acctctaaca atgaaattgt gtagattcaa aaatagaatt aattcgtaat aaaaagtcga
     3901 aaaaaattgt gctccctccc cccattaata ataattctat cccaaaatct acacaatgtt
     3961 ctgtgtacac ttcttatgtt ttttacttct gataaatttt tttgaaacat catagaaaaa
     4021 accgcacaca aaatacctta tcatatgtta cgtttcagtt tatgaccgca atttttattt
     4081 cttcgcacgt ctgggcctct catgacgtca aatcatgctc atcgtgaaaa agttttggag
     4141 tatttttgga atttttcaat caagtgaaag tttatgaaat taattttcct gcttttgctt
     4201 tttggggttt cccctattgt ttgtcaagat ttcgaggacg gcgtttttct tgctaaaatc
     4261 acaagtattg atgagcacga tgcaagaaag atcggaagaa ggtttgggtt tgaggctcag
     4321 tggaaggtga gtagaagttg ataatttgaa agtggagtag tgtctatggg gtttttgccg
     4381 agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     4441 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4501 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4561 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4621 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4681 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4741 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4801 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4861 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4921 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4981 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5041 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5101 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5161 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5221 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5281 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5341 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5401 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5461 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5521 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5581 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5641 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5701 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5761 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5821 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5881 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5941 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6001 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6061 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6121 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6181 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6241 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6301 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6361 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6421 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6481 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6541 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
//
