LOCUS       sequence-72953-e        4468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for Golden Gate assembly; middle entry vector for
            Gateway cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4468)
  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 4468)
  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: pME_GGWDest+
FEATURES             Location/Qualifiers
     source          1..4468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        618..636
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     protein_bind    660..759
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     761..777
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        787..805
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      820..829
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(842..1225)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSYLGDTIEYSSYASNALGALPYGRPAGGREFTSDIEFPR
                     PPWRPGACDVGPNSPYSESYYNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEA
                     RTDRPSQQLRSLNGEWTRPVAAH"
     primer_bind     991..1013
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1006..1022
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1029..1047
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1078..1087
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1082..1091
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1185..1203)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1221..1237)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1234..1256)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1245..1261
                     /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(1269..1275,1276..1293,1294..1299))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1314..1335
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     1491..1507
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1492..1508
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1542..1558)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1542..1558)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1595..1615)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1595..1613)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(1642..1741)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     intron          1816..2388
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_signal    2461..2595
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2485..2504)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2539..2558
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(2710..2728)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2733..2749)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2851..2870)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2862..3671
                     /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(2929..2948)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3818..4406
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4307..4326
                     /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 gcagggcgcc
      181 tcccccattg acgtcaatta cggtaaatgg cccgcctggc tcaatgccca ttgacgtcaa
      241 taggaccacc caccattgac gtcaatggga tggctcattg cccattcata tccgttctca
      301 cgccccctat tgacgtcaat gacggtaaat ggcccacttg gcagtacatc aatatctatt
      361 aatagtaact tggcaagtac attactattg gaagtacgcc agggtacatt ggcagtactc
      421 ccattgacgt caatggcggt aaatggcccg cgatggctgc caagtacatc cccattgacg
      481 tcaatgggga ggggcaatga cgcaaatggg cgttccattg acgtaaatgg gcggtaggcg
      541 tgcctaatgg gaggtctata taagcaatgc tcgtttaggg aaccgccatt ctgcctgggg
      601 acgtcggagc aagcttgatt taggtgacac tatagacggc cagtcttaag ctcgggcccc
      661 aaataatgat tttattttga ctgatagtga cctgttcgtt gcaacaaatt gatgagcaat
      721 gcttttttat aatgccaact ttgtacaaaa aagcaggctt gtaaaacgac ggccagtgag
      781 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ccgccatggc gagacccgcg
      841 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
      901 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      961 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     1021 gtgaattgta atacgactca ctatagggcg aattgggccc gacgtcgcat gctcccggcc
     1081 gccatggcgg ccgcgggaat tcgatatcac tagtgaattc gcggccgcct gcaggtcgac
     1141 catatgggag agctcccaac gcgttggatg catagcttga gtattctata gtgtcaccta
     1201 aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     1261 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     1321 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     1381 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     1441 tcttccgctt cctcgctcac tgactcgctg cgctcggggt ctcttaaggc tcgaggtcga
     1501 cggtatcgat aagcttgata tcgaattcct gcagcccggg ggatccacta gttctagagc
     1561 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     1621 tggcgtaatc atggtcatag cacccagctt tcttgtacaa agttggcatt ataagaaagc
     1681 attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt
     1741 gccatccagc tgatatcccc tctcatcctc ccgggtaact aagtaaggat ctcgaccgat
     1801 cctgagaact tcagggtgag tttggggacc cttgattgtt ctttcttttt cgctattgta
     1861 aaattcatgt tatatggagg gggcaaagtt ttcagggtgt tgtttagaat gggaagatgt
     1921 cccttgtatc accatggacc ctcatgataa ttttgtttct ttcactttct actctgttga
     1981 caaccattgt ctcctcttat tttcttttca ttttctgtaa ctttttcgtt aaactttagc
     2041 ttgcatttgt aacgaatttt taaattcact tttgtttatt tgtcagattg taagtacttt
     2101 ctctaatcac ttttttttca aggcaatcag ggtatattat attgtacttc agcacagttt
     2161 tagagaacaa ttgttataat taaatgataa ggtagaatat ttctgcatat aaattctggc
     2221 tggcgtggaa atattcttat tggtagaaac aactacaccc tggtcatcat cctgcctttc
     2281 tctttatggt tacaatgata tacactgttt gagatgagga taaaatactc tgagtccaaa
     2341 ccgggcccct ctgctaacca tgttcatgcc ttcttctctt tcctacagct cctgggcaac
     2401 gtgctggttg ttgtgctgtc tcatcatttt ggcaaagaat taattcctcg acggatcgta
     2461 gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     2521 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     2581 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     2641 gtgtgggagg ttttttaatt cgcggccgct ctagaactca tatggcgcgc cagatctggc
     2701 gtgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc
     2761 ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     2821 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     2881 aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     2941 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg
     3001 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     3061 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     3121 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg
     3181 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     3241 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     3301 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     3361 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     3421 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     3481 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     3541 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     3601 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     3661 agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt
     3721 gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg tcgttccact
     3781 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3841 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3901 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3961 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4021 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4081 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4141 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4201 agcgtgagca ttgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4261 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4321 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4381 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4441 ccttttgctg gccttttgct cacatgtt
//
