LOCUS       sequence-72952-e        4463 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 4463)
  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 4463)
  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..4463
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        618..636
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     protein_bind    661..760
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     762..778
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        788..806
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(837..1220)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSYLGDTIEYSSYASNALGALPYGRPAGGREFTSDIEFPR
                     PPWRPGACDVGPNSPYSESYYNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEA
                     RTDRPSQQLRSLNGEWTRPVAAH"
     primer_bind     986..1008
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1001..1017
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1024..1042
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1073..1082
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1077..1086
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1180..1198)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1216..1232)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1229..1251)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1240..1256
                     /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(1264..1270,1271..1288,1289..1294))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1309..1330
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     1486..1502
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1487..1503
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1537..1553)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1537..1553)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1590..1610)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1590..1608)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(1637..1736)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     intron          1811..2383
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_signal    2456..2590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2480..2499)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2534..2553
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(2705..2723)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2728..2744)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2846..2865)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2857..3666
                     /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(2924..2943)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3813..4401
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4302..4321
                     /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 tatagaacgg ccagtcttaa gctcgggccc
      661 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
      721 tgctttttta taatgccaac tttgtacaaa aaagcaggct tgtaaaacga cggccagtga
      781 gcgcgcgtaa tacgactcac tatagggcga attgggtacc gtatcgagac ccgcgcttaa
      841 tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
      901 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
      961 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa
     1021 ttgtaatacg actcactata gggcgaattg ggcccgacgt cgcatgctcc cggccgccat
     1081 ggcggccgcg ggaattcgat atcactagtg aattcgcggc cgcctgcagg tcgaccatat
     1141 gggagagctc ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc acctaaatag
     1201 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     1261 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     1321 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     1381 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     1441 cgcttcctcg ctcactgact cgctgcgctc ggggtctctt aaggctcgag gtcgacggta
     1501 tcgataagct tgatatcgaa ttcctgcagc ccgggggatc cactagttct agagcggccg
     1561 ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     1621 taatcatggt catagcaccc agctttcttg tacaaagttg gcattataag aaagcattgc
     1681 ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat
     1741 ccagctgata tcccctctca tcctcccggg taactaagta aggatctcga ccgatcctga
     1801 gaacttcagg gtgagtttgg ggacccttga ttgttctttc tttttcgcta ttgtaaaatt
     1861 catgttatat ggagggggca aagttttcag ggtgttgttt agaatgggaa gatgtccctt
     1921 gtatcaccat ggaccctcat gataattttg tttctttcac tttctactct gttgacaacc
     1981 attgtctcct cttattttct tttcattttc tgtaactttt tcgttaaact ttagcttgca
     2041 tttgtaacga atttttaaat tcacttttgt ttatttgtca gattgtaagt actttctcta
     2101 atcacttttt tttcaaggca atcagggtat attatattgt acttcagcac agttttagag
     2161 aacaattgtt ataattaaat gataaggtag aatatttctg catataaatt ctggctggcg
     2221 tggaaatatt cttattggta gaaacaacta caccctggtc atcatcctgc ctttctcttt
     2281 atggttacaa tgatatacac tgtttgagat gaggataaaa tactctgagt ccaaaccggg
     2341 cccctctgct aaccatgttc atgccttctt ctctttccta cagctcctgg gcaacgtgct
     2401 ggttgttgtg ctgtctcatc attttggcaa agaattaatt cctcgacgga tcgtagatcc
     2461 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     2521 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     2581 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     2641 ggaggttttt taattcgcgg ccgctctaga actcatatgg cgcgccagat ctggcgtgat
     2701 atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct ctggcccgtg
     2761 tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa
     2821 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     2881 tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc
     2941 gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca
     3001 gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc
     3061 agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact
     3121 cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt ccaggtatta
     3181 gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg
     3241 ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct
     3301 caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt
     3361 aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg
     3421 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     3481 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     3541 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     3601 tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     3661 ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg
     3721 gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg
     3781 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3841 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3901 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3961 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4021 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4081 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4141 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4201 gagcattgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4261 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4321 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4381 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4441 tgctggcctt ttgctcacat gtt
//
