LOCUS       sequence-72951-e        3348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for Golden Gate assembly; 5' entry vector for 
            Gateway cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3348)
  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 3348)
  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: p5E_GGWDest+
FEATURES             Location/Qualifiers
     source          1..3348
                     /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"
     protein_bind    593..688
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      706..715
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(728..1111)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSYLGDTIEYSSYASNALGALPYGRPAGGREFTSDIEFPR
                     PPWRPGACDVGPNSPYSESYYNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEA
                     RTDRPSQQLRSLNGEWTRPVAAH"
     primer_bind     892..908
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        915..933
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      964..973
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      968..977
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1071..1089)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1072..1089)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1107..1123)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1120..1142)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1131..1147
                     /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(1155..1161,1162..1179,1180..1185))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1200..1221
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     1377..1393
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     protein_bind    1394..1517
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(1590..1608)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1613..1629)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1731..1750)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1742..2551
                     /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(1809..1828)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2698..3286
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3187..3206
                     /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 tcgggcccga gttaacgcta ccatggagct ccaaataatg
      601 attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgataagca atgctttttt
      661 ataatgccaa ctttgtatag aaaagttgag ggcgaattgg gtaccgccgc catggcgaga
      721 cccgcgctta atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt
      781 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
      841 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
      901 cggccagtga attgtaatac gactcactat agggcgaatt gggcccgacg tcgcatgctc
      961 ccggccgcca tggcggccgc gggaattcga tatcactagt gaattcgcgg ccgcctgcag
     1021 gtcgaccata tgggagagct cccaacgcgt tggatgcata gcttgagtat tctatagtgt
     1081 cacctaaata gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     1141 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     1201 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1261 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1321 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggggtctct taaggctcga
     1381 ggtcgacggt atccaagttt gtacaaaaaa gttgaacgag aaacgtaaaa tgatataaat
     1441 atcaatatat taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa
     1501 catatgcagt cactatgaac caactactta gatggtatta gtgacctgta gaattcgagc
     1561 tctagagctg cagggcggcc gcgatatccc ctatagtgag tcgtattaca tggtcatagc
     1621 tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa
     1681 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     1741 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     1801 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     1861 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     1921 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     1981 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     2041 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     2101 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     2161 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     2221 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     2281 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     2341 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     2401 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     2461 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     2521 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg
     2581 gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt
     2641 gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2701 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2761 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2821 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     2881 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     2941 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3001 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3061 caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag
     3121 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3181 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3241 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3301 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtt
//
