LOCUS       sequence-322107-        6064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway (attR4-attR2) vector for expression of multiple 
            proteins using the Bac-to-Bac insect cell system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6064)
  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 6064)
  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-624 (attR4-attR2)
FEATURES             Location/Qualifiers
     source          1..6064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    2..125
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(150..155,156..173,174..180)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             234..893
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(300..319)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1214..1519
                     /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     1398..1417
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1560..1684)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    1826..1960
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1863..1882)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1917..1936
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  1989..2154
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      2338..2792
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2425..2444)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2634..2655
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2818..2922
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2923..2991,2992..3783)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3141..3160)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3954..4542
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4443..4462
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4696..4713
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  4847..5071
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(5138..5671)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(5841..5846,5847..5863,5864..5869))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 acaactttgt atagaaaagt tgaacgagaa acgtaaaatg atataaatat caatatatta
       61 aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca
      121 ctatggcggc cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg
      181 tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga
      241 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
      301 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
      361 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
      421 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
      481 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
      541 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
      601 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
      661 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
      721 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
      781 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
      841 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taaacgcgtg
      901 gatccggctt actaaaagcc agataacagt atgcgtattt gcgcgcaccg gtgctagcgt
      961 atacccgaag tatgtcaaaa agaggtatgc tatgaagcag cgtattacag tgacagttga
     1021 cagcgacagc tatcagttgc tcaaggcata tatgatgtca atatctccgg tctggtaagc
     1081 acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag
     1141 gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc tgacgagaac
     1201 aggggctggt gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct
     1261 gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct
     1321 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat
     1381 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat
     1441 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct
     1501 gatgttctgg ggaatataaa tgtcaggctc ccttatacac agccagtctg caggtcgacc
     1561 atagtgactg gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct
     1621 aatttaatat attgatattt atatcatttt acgtttctcg ttcaactttc ttgtacaaag
     1681 ttgtgatcga gctagcaagc ttgtcgagaa gtactagagg atcataatca gccataccac
     1741 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     1801 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     1861 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     1921 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tgatcactgc ttgagcctag
     1981 gagatccgaa ccagataagt gaaatctagt tccaaactat tttgtcattt ttaattttcg
     2041 tattagctta cgacgctaca cccagttccc atctattttg tcactcttcc ctaaataatc
     2101 cttaaaaact ccatttccac ccctcccagt tcccaactat tttgtccgcc cacagcgggg
     2161 catttttctt cctgttatgt ttttaatcaa acatcctgcc aactccatgt gacaaaccgt
     2221 catcttcggc tactttttct ctgtcacaga atgaaaattt ttctgtcatc tcttcgttat
     2281 taatgtttgt aattgactga atatcaacgc ttatttgcag cctgaatggc gaatgggacg
     2341 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     2401 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     2461 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2521 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     2581 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttctta atagtggact
     2641 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2701 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2761 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     2821 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     2881 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     2941 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3001 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3061 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3121 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     3181 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3241 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3301 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3361 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     3421 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     3481 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3541 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3601 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     3661 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3721 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     3781 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     3841 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3901 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3961 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4021 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4081 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4141 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4201 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4261 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4321 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4381 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4441 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4501 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4561 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4621 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4681 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4741 tttctcctta cgcatctgtg cggtatttca caccgcatag accagccgcg taacctggca
     4801 aaatcggtta cggttgagta ataaatggat gccctgcgta agcgggtgtg ggcggacaat
     4861 aaagtcttaa actgaacaaa atagatctaa actatgacaa taaagtctta aactagacag
     4921 aatagttgta aactgaaatc agtccagtta tgctgtgaaa aagcatactg gacttttgtt
     4981 atggctaaag caaactcttc attttctgaa gtgcaaattg cccgtcgtat taaagagggg
     5041 cgtggccaag ggcatggtaa agactatatt cgcggcgttg tgacaattta ccgaacaact
     5101 ccgcggccgg gaagccgatc tcggcttgaa cgaattgtta ggtggcggta cttgggtcga
     5161 tatcaaagtg catcacttct tcccgtatgc ccaactttgt atagagagcc actgcgggat
     5221 cgtcaccgta atctgcttgc acgtagatca cataagcacc aagcgcgttg gcctcatgct
     5281 tgaggagatt gatgagcgcg gtggcaatgc cctgcctccg gtgctcgccg gagactgcga
     5341 gatcatagat atagatctca ctacgcggct gctcaaactt gggcagaacg taagccgcga
     5401 gagcgccaac aaccgcttct tggtcgaagg cagcaagcgc gatgaatgtc ttactacgga
     5461 gcaagttccc gaggtaatcg gagtccggct gatgttggga gtaggtggct acgtctccga
     5521 actcacgacc gaaaagatca agagcagccc gcatggattt gacttggtca gggccgagcc
     5581 tacatgtgcg aatgatgccc atacttgagc cacctaactt tgttttaggg cgactgccct
     5641 gctgcgtaac atcgttgctg ctccataaca tcaaacatcg acccacggcg taacgcgctt
     5701 gctgcttgga tgcccgaggc atagactgta caaaaaaaca gtcataacaa gccatgaaaa
     5761 ccgccactgc gccgttacca ccgctgcgtt cggtcaaggt tctggaccag ttgcgtgagc
     5821 gcatacgcta cttgcattac agtttacgaa ccgaacaggc ttatgtcaac tgggttcgtg
     5881 ccttcatccg tttccacggt gtgcgtcacc cggcaacctt gggcagcagc gaagtcgagg
     5941 catttctgtc ctggctggcg aacgagcgca aggtttcggt ctccacgcat cgtcaggcat
     6001 tggcggcctt gctgttcttc tacggcaagg tgctgtgcac ggatctacca tgggatcctc
     6061 gatc
//
