LOCUS       sequence-322139-        6634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway (attR4-attR2) vector for expression of multiple 
            proteins in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6634)
  AUTHORS   Wall VE, Garvey LA, Mehalko JL, Procter LV, Esposito D
  TITLE     Combinatorial assembly of clone libraries using site-specific 
            recombination.
  JOURNAL   Methods Mol Biol. 2014;1116:193-208. doi: 
            10.1007/978-1-62703-764-8_14.
  PUBMED    24395366
REFERENCE   2  (bases 1 to 6634)
  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: "Methods Mol
            Biol."; date: "2014"; volume: "1116:193-208. doi"; pages: " 
            10.1007/978-1-62703-764-8_14"
COMMENT     Sequence Label: pDest-894 (attR4-attR2)
FEATURES             Location/Qualifiers
     source          1..6634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     590..609
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(629..706)
                     /gene="lacI"
                     /label=lacI promoter
     protein_bind    807..930
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(955..960,961..978,979..985)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1039..1698
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1105..1124)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2019..2324
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2203..2222
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2365..2489)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2513..2530
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2583..2601)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2597..2644
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2681..3136
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2768..2787)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2978..2999
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3228..4043)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3228..4043)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3951..3970
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4035..4054
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4165..4753
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4654..4673
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4907..4924
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4939..5078
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5004..5023)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5164..5186
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5180..5371)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6180..6634,1..628))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
       61 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
      121 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
      181 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
      241 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
      301 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
      361 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
      421 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
      481 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
      541 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
      601 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
      661 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
      721 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
      781 accgccgccg caaggaatgg tgatcacaac tttgtataga aaagttgaac gagaaacgta
      841 aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata
      901 ctgtaaaaca caacatatcc agtcactatg gcggccgcat taggcacccc aggctttaca
      961 ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatccgtc gagattttca
     1021 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc
     1081 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac
     1141 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     1201 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt
     1261 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     1321 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     1381 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     1441 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     1501 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc
     1561 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
     1621 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     1681 tggcagggcg gggcgtaaac gcgtggatcc ggcttactaa aagccagata acagtatgcg
     1741 tatttgcgcg caccggtgct agcgtatacc cgaagtatgt caaaaagagg tatgctatga
     1801 agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga
     1861 tgtcaatatc tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc
     1921 cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat
     1981 gaacggctct tttgctgacg agaacagggg ctggtgaaat gcagtttaag gtttacacct
     2041 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc
     2101 ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc
     2161 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata
     2221 tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa
     2281 atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta
     2341 tacacagcca gtctgcaggt cgaccatagt gactggatat gttgtgtttt acagtattat
     2401 gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt
     2461 tctcgttcaa ctttcttgta caaagttgtg atagcttgcg gccgcactcg agcaccacca
     2521 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     2581 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     2641 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     2701 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2761 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2821 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2881 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2941 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     3001 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     3061 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     3121 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3181 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     3241 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     3301 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     3361 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     3421 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     3481 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     3541 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     3601 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     3661 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     3721 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     3781 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     3841 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     3901 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     3961 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     4021 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     4081 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4141 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4201 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4261 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4321 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4381 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4441 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4501 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4561 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4621 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4681 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4741 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4801 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4861 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4921 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4981 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     5041 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     5101 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5161 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     5221 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     5281 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     5341 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     5401 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     5461 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     5521 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     5581 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     5641 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     5701 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     5761 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     5821 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     5881 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     5941 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     6001 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     6061 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     6121 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     6181 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6241 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     6301 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     6361 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     6421 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     6481 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     6541 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     6601 ctccagtcgc cttcccgttc cgctatcggc tgaa
//
