LOCUS       sequence-483112-        6276 bp    DNA     circular SYN 19-AUG-2025
DEFINITION  Allows for tiam-1::mGFP entry into Gateway Vectors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6276)
  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 6276)
  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: pBD28
FEATURES             Location/Qualifiers
     source          1..6276
                     /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             join(3406..3408,3409..3411,3412..4125)
                     /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(3451..3472)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3712..3731)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4059..4080
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4155..4254)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4271..4290)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4272..4290)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4295..4311)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4295..4311)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4742..5533
                     /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      5626..6214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6115..6134
                     /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 catatgggta ccatgggctc acgcctctca
      721 tgttcttgct cacaagccga atgctggacg aatgatgaat ctacattgag aattgacgcg
      781 gagttgtttg agttatcaca ggacggacaa aagtgggata agcttgacgg aaggctggcc
      841 aacgtacgtg tattcaacgc attcgacgac tcttccccac gtcttcttgc cacatcatca
      901 tccggtcaag ttcttctgga cactctaatt ccaatgggag aaaaggttca taaagtcagc
      961 gacttttttg tgtatttgaa gacggaagga agaacaattg gattcaatac gttaagctcg
     1021 agggacactt ccttactcat ttctcaggcg acaaatacaa ctgccttcaa tatgtttcaa
     1081 cattcattct cagtttccgt atccagaatt gctacatttg aaggtgattc tattcaacct
     1141 tgtgatgagc agccagaaac gtcaaatgtc tcactaaatc cgttaaatat ggttttcaac
     1201 tacaaaggat cacctgttga gattgatttg ctggaatgtt tcgcgtgctc atcagaagct
     1261 gaaaactgtg tcgacatagc acatgccaac aacatatttc gtgtaattgt gtcagcacat
     1321 gctcatcttt ttcttattca agctctgaat gtatcatcac ttcttcttgc tcgaaccacc
     1381 tcgaatctag ttgccatcaa ttatttggct cggcaaaatg ataaaattca acaaaaattg
     1441 attgctctgt tggaaacatc agaatcaagt gagaatgcgt cgaaggaaga tattcgacaa
     1501 atgcttcttc tgaagaatgt agtagtgaca cagagattga actcacttca acagagagca
     1561 ctaccgggaa ttgagcttat ccaacacgat atctcacctc aaattgatca gaagctactc
     1621 gaaaagtgtc gacttccatc actgttttta tatcatttta atatgaattc gtttcgaata
     1681 tggcgaaaac caatggaaac acgtgacatg aacacaatgg gatcatcaat ggcatccacc
     1741 agtcaatacc atcagattcc gattcaaatc cagaatcaga atccgttacc gtctcaacag
     1801 attcagccac aacaacaaca acaataccat ccaccagatc atgtgacaac tcataatggt
     1861 acaactacgt ttcatactac gaatcccaca aaaccaagac tttgtgaagg atcgttggct
     1921 cctggatgct cattggccga ggcatctgaa cagagaaaac gagagatggc ggagcagaaa
     1981 aaagagttgg caggcccatc ttccgcagag gattacttcg tcagaaagac aaatggacgt
     2041 cttggactca ctatatacgc gcataacgat gatggggtca tcagggctga ggttcgtgga
     2101 gtgacgtcat ttgctccgag atgtgctcaa gttggagatt cggttgtagc ggtagacagt
     2161 gaactcatat catcagttag aaacgcttcg gatgttgaga aacttttgag aatcggaaaa
     2221 gtaatacatt tacgaagaaa gactcctcta actccaagag caccgtctca actcgacgca
     2281 actaatgtca ataagaaaac gggctgtgca aagttggcga tggctctgca agaattgttg
     2341 gtcactgaga agaaatatgt cagcgatctt cgagagatga atgaagtttt tctgtgtatg
     2401 agacaagtcc gagatattat gaatgcagca atacggttgt ataaaatgca aacatcgttt
     2461 gtggacagta ttgaggaagc cattggagac atgagtagac aggatatttc tgttgctcag
     2521 attagggatt ctgtaatgcg agtgtgtgca gtattcatca acaaatgttc tgatttcaaa
     2581 atctatgcag aatatgcagc cggttaccat ctcctccaac atgagatcaa aaacaaaaaa
     2641 gagcttctat caaaattgga agctgtaaac agtacaagag agcaacattg ttcttgggaa
     2701 tctcggatga ttaaaccagt ccagagaatt gtacaatatc cacttttact gaaaaacatt
     2761 gccgatgcac ttccgaagga tgcacgagag agggttcaag tggaggcagc attgcaaaaa
     2821 atgcagacgt ctgcagaata tgtgaatgaa atgcaaaggc ttcatgaaga ttatgcacaa
     2881 tatatggaga ctgttaggaa agctaatgag ccaatgttgg ctgaaaagag tcttcgcctc
     2941 gacactcgag agcttctagt ttttgcacac ataaaatgga gagatgctcc agcggagcac
     3001 tacgttgtgt tcgtgtttca ctctttaata ctgctccttc catcgtatgc taggaaagag
     3061 attaagatga aatggactcg tgtcctccca ataaatgaag ttgatatcaa cgaaatgcca
     3121 aatgatgcat taaatctaaa actttatcat gctgcttttg aaggaccaaa tggacaatta
     3181 agtcattcca atccaaatac agtctacaat attgaatgtt gtcaatctca actgaaacag
     3241 catcttatca aaaatatcaa aaaagctcga actttattct ctcgagaatc acatcgtcca
     3301 ttaagtggat ctagtcaatc ggatggtggt tatgtgtcag aagtctctaa ggaacatcga
     3361 aattcaaaag gtaccagtgg ttccggagga tctggaggat ctggtatggt gagcaagggc
     3421 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     3481 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     3541 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     3601 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     3661 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     3721 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     3781 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     3841 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     3901 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     3961 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     4021 tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     4081 accgccgccg ggatcactct cggcatggac gagctgtaca agtagcatat ggcggccgcg
     4141 gaagggcgaa ttcgacccag ctttcttgta caaagttggc attataaaaa ataattgctc
     4201 atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttgccatcc
     4261 agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct ggcagctctg
     4321 gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat catcatgcct
     4381 cctctagacc agccaggaca gaaatgcctc gacttcgctg ctgcccaagg ttgccgggtg
     4441 acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct gttcggttcg
     4501 taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg
     4561 cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt
     4621 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     4681 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     4741 catgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     4801 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     4861 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     4921 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     4981 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     5041 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     5101 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     5161 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     5221 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     5281 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     5341 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     5401 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     5461 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt
     5521 agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc
     5581 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5641 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5701 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5761 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5821 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5881 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5941 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6001 atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6061 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6121 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6181 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6241 gttcctggcc ttttgctggc cttttgctca catgtt
//
