LOCUS       sequence-483117-        6593 bp    DNA     circular SYN 19-AUG-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 6593)
  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 6593)
  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: pREA16
FEATURES             Location/Qualifiers
     source          1..6593
                     /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    1300..1324
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2145..2210
                     /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(2853..2855,2856..2858,2859..3572)
                     /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(2898..2919)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3159..3178)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3506..3527
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3602..3626)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3652..3668)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3652..3668)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4405..4425)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4405..4423)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4444..4460)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4444..4460)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4457..4479)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4468..4484
                     /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(4492..4498,4499..4516,4517..4522))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4537..4558
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4675..4692)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4846..5434)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4926..4945)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5605..6396,6397..6465))
                     /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     6228..6247
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6466..6570)
                     /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 tctagcgcca
      781 aggcaatctt cgtaaaaaat aacaatagag tcatccctca gaacagaaaa gaaacccgcc
      841 cgggggctca aatacacatg tcatttttct tttttcgcct gatttttagc ctaaattagt
      901 gtttagcaat tttttaaggt aactcggtgg agtcatttga actttttaaa ctaaaatttc
      961 agaaaaaatt cttttttttt gtcaaaaaat aattttcccg ccggaatttt tcaaaaaaat
     1021 aatttttatt ttatttttca agttccttgt ttcctcttta tttttttatt ttgcccccaa
     1081 aagttttaca ctaaaatatt taattttcag gtaattcatc gaaagtcttt ctattttccg
     1141 catctcttgt tcaaaagctt gcatgcctgc aggtcgactc tagaggatcc ccgggattgg
     1201 ccaaaggacc caaaggtatg tttcgaatga tactaacata acatagaaca ttttcaggag
     1261 gacccttgga gggtaccggt agaaaaaaag cttgatatca caagtttgta caaaaaagca
     1321 ggctccgaat tcgcccttgg actagtcata tgatggtctc aaagggtgaa gaagataaca
     1381 tggcaattat taaagagttt atgcgtttca aggtgcatat ggagggatct gtcaatgggc
     1441 atgagtttga aattgaaggt gaaggagaag gccgaccata tgagggaaca caaaccgcaa
     1501 aactaaaggt aactaaaggc ggaccattac cattcgcctg ggacatcctc tctccacagt
     1561 tcatgtatgg aagtaaagct tatgttaaac atccggcaga tataccagat tatttgaaac
     1621 tttcattccc ggagggtttt aagtgggaac gcgtaatgaa ttttgaagac ggaggagttg
     1681 ttacagtgac gcaagactca agcctccaag atggagaatt tatttataaa gtcaaacttc
     1741 gaggaacgaa tttcccctcg gatggacctg ttatgcagaa gaagactatg ggatgggaag
     1801 cttcaagtga aagaatgtac cctgaagacg gtgctcttaa gggagagatt aaacaacgtc
     1861 ttaaattgaa agatggagga cattacgatg ctgaggtgaa gacaacttac aaagccaaaa
     1921 aaccagttca gctgccagga gcgtacaatg ttaatattaa actggatatc acctcccaca
     1981 acgaggatta cactatcgtt gagcaatatg aaagagctga agggcggcac tcgacaggtg
     2041 gcatggatga attgtataag actagtggtt ccggaggatc tggaggatct ggtaccatct
     2101 ctcgaccgag caatttcgaa cacacaattc atgtcggagg taccgtaaaa caaacgctca
     2161 actttgactt acttaaactt gcgggcgacg ttgagtcaaa cccaggacca ggatccatgc
     2221 aagcaatcaa atgtgtcgtc gttggtgacg gagccgtcgg taagacgtgt ctcctgctat
     2281 cgtacactac aaacgctttt cccggagaat atattctaac ggtattcgac acctactcaa
     2341 caaatgtgat ggtcgacgga aggccaataa atctcagcct atgggacaca gctggacagg
     2401 acgattacga tcaattccgc cacctgtcat ttccacaaac agacgtattc ctcgtatgct
     2461 ttgcactgaa taatccagca agttttgaga atgttcgtgc aaaatggtat ccagaagtat
     2521 cacatcattg cccaaataca ccgattattt tggttggaac taaagctgat ttacgcgagg
     2581 atcgagatac tattgaacgg ctccgtgaac gtcggctcca accagtgagc cacacccagg
     2641 gttacgtgat ggcaaaggaa atcaaggcgg tcaagtacct ggaatgctcg gcgcttaccc
     2701 aaattggatt gaaacaagtt ttcgatgagg caattcgtac tgggctcacc ccgccacaaa
     2761 caccacaaac gagagccaaa aagagcaatt gcacggtgct tggtggttcc ggtggatccg
     2821 gttccggagg atctggagga tctgcggccg cgatggtgag caagggcgag gagctgttca
     2881 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     2941 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     3001 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     3061 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     3121 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     3181 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     3241 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     3301 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     3361 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     3421 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca
     3481 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     3541 tcactctcgg catggacgag ctgtacaagt agcatatggc ggccgcggaa gggcgaattc
     3601 gacccagctt tcttgtacaa agtggtgata tcgaattcct gcagcccggg ggatccacta
     3661 gttctagagc ggccgccacc gcggccgctg tcatcagatc gccatctcgc gcccgtgcct
     3721 ctgacttcta agtccaatta ctcttcaaca tccctacatg ctctttctcc ctgtgctccc
     3781 accccctatt tttgttatta tcaaaaaact tctcttaatt tctttgtttt ttagcttctt
     3841 ttaagtcacc tctaacaatg aaattgtgta gattcaaaaa tagaattaat tcgtaataaa
     3901 aagtcgaaaa aaattgtgct ccctcccccc attaataata attctatccc aaaatctaca
     3961 caatgttctg tgtacacttc ttatgttttt tacttctgat aaattttttt gaaacatcat
     4021 agaaaaaacc gcacacaaaa taccttatca tatgttacgt ttcagtttat gaccgcaatt
     4081 tttatttctt cgcacgtctg ggcctctcat gacgtcaaat catgctcatc gtgaaaaagt
     4141 tttggagtat ttttggaatt tttcaatcaa gtgaaagttt atgaaattaa ttttcctgct
     4201 tttgcttttt ggggtttccc ctattgtttg tcaagatttc gaggacggcg tttttcttgc
     4261 taaaatcaca agtattgatg agcacgatgc aagaaagatc ggaagaaggt ttgggtttga
     4321 ggctcagtgg aaggtgagta gaagttgata atttgaaagt ggagtagtgt ctatggggtt
     4381 tttgccgagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     4441 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4501 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4561 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4621 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4681 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4741 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     4801 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     4861 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4921 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4981 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5041 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5101 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5161 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5221 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5281 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5341 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5401 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5461 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5521 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5581 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5641 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5701 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5761 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5821 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5881 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5941 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6001 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6061 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6121 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6181 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6241 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6301 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6361 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6421 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6481 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6541 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac
//
