LOCUS       sequence-82571-e        3337 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for Golden Gate assembly; 3' entry vector for 
            Gateway cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3337)
  AUTHORS   Kirchmaier S, Lust K, Wittbrodt J
  TITLE     Golden GATEway cloning--a combinatorial approach to generate fusion 
            and recombination constructs.
  JOURNAL   PLoS One. 2013 Oct 7;8(10):e76117. doi: 
            10.1371/journal.pone.0076117.
  PUBMED    24116091
REFERENCE   2  (bases 1 to 3337)
  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"; journalName: "PLoS One.";
            date: "2013-10-7"; volume: "8(10):e76117. doi"; pages: " 
            10.1371/journal.pone.0076117"
COMMENT     Sequence Label: p3E_GGWDest-
FEATURES             Location/Qualifiers
     source          1..3337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(780..1163)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSYLGDTIEYSSYASNALGALPYGRPAGGREFTSDIEFPR
                     PPWRPGACDVGPNSPYSESYYNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEA
                     RTDRPSQQLRSLNGEWTRPVAAH"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        967..985
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1016..1025
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1020..1029
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1123..1141)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1124..1141)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1159..1175)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1172..1194)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1183..1199
                     /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(1207..1213,1214..1231,1232..1237))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1252..1273
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     1429..1445
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     protein_bind    complement(1446..1541)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1579..1597)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1602..1618)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1720..1739)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1731..2540
                     /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(1798..1817)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2687..3275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3176..3195
                     /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 tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtgaggg cgaattgggt accgtatcga gacccgcgct
      781 taatgcgccg ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg
      841 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
      901 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
      961 gaattgtaat acgactcact atagggcgaa ttgggcccga cgtcgcatgc tcccggccgc
     1021 catggcggcc gcgggaattc gatatcacta gtgaattcgc ggccgcctgc aggtcgacca
     1081 tatgggagag ctcccaacgc gttggatgca tagcttgagt attctatagt gtcacctaaa
     1141 tagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     1201 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     1261 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     1321 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     1381 ttccgcttcc tcgctcactg actcgctgcg ctcggggtct cttaaggctc gaggtcgacg
     1441 gtatccaact ttattataca aagttggcat tataaaaaag cattgcttat caatttgttg
     1501 caacgaacag gtcactatca gtcaaaataa aatcattatt tggagctcca tggtagcgtt
     1561 aacgcggccg cgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc
     1621 agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat
     1681 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     1741 ttcaacggga aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     1801 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga
     1861 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     1921 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     1981 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag
     2041 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     2101 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     2161 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     2221 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt
     2281 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     2341 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     2401 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     2461 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     2521 tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta
     2581 cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt
     2641 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2701 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2761 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2821 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2881 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2941 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3001 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3061 gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3121 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3181 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3241 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3301 ggttcctggc cttttgctgg ccttttgctc acatgtt
//
