LOCUS       sequence-327081-        6080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway (attR4-attR3) 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 6080)
  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 6080)
  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-623 (attR4-attR3)
FEATURES             Location/Qualifiers
     source          1..6080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    129..263
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(166..185)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     220..239
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  292..457
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      641..1095
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(728..747)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     937..958
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1121..1225
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1226..1294,1295..2086)
                     /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(1444..1463)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2257..2845
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2746..2765
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2999..3016
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  3150..3374
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(3441..3974)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(4144..4149,4150..4166,4167..4172))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    4367..4490
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(4531..4836)
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(4633..4652)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(5156..5836)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     5751..5770
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(5890..5896,5897..5914,5915..5920))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(5945..6068)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 agcttgtcga gaagtactag aggatcataa tcagccatac cacatttgta gaggttttac
       61 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
      121 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
      181 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
      241 atgtatctta tcatgtctgg atctgatcac tgcttgagcc taggagatcc gaaccagata
      301 agtgaaatct agttccaaac tattttgtca tttttaattt tcgtattagc ttacgacgct
      361 acacccagtt cccatctatt ttgtcactct tccctaaata atccttaaaa actccatttc
      421 cacccctccc agttcccaac tattttgtcc gcccacagcg gggcattttt cttcctgtta
      481 tgtttttaat caaacatcct gccaactcca tgtgacaaac cgtcatcttc ggctactttt
      541 tctctgtcac agaatgaaaa tttttctgtc atctcttcgt tattaatgtt tgtaattgac
      601 tgaatatcaa cgcttatttg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
      661 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
      721 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
      781 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
      841 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
      901 tttcgccctt tgacgttgga gtccacgttc ttaatagtgg actcttgttc caaactggaa
      961 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1021 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1081 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1141 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1201 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1261 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1321 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1381 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1441 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     1501 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     1561 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     1621 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     1681 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     1741 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     1801 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     1861 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     1921 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     1981 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2041 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2101 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2161 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2221 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2281 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2341 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2401 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2461 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2521 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2581 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2641 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2701 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     2761 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     2821 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     2881 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     2941 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3001 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3061 gtgcggtatt tcacaccgca tagaccagcc gcgtaacctg gcaaaatcgg ttacggttga
     3121 gtaataaatg gatgccctgc gtaagcgggt gtgggcggac aataaagtct taaactgaac
     3181 aaaatagatc taaactatga caataaagtc ttaaactaga cagaatagtt gtaaactgaa
     3241 atcagtccag ttatgctgtg aaaaagcata ctggactttt gttatggcta aagcaaactc
     3301 ttcattttct gaagtgcaaa ttgcccgtcg tattaaagag gggcgtggcc aagggcatgg
     3361 taaagactat attcgcggcg ttgtgacaat ttaccgaaca actccgcggc cgggaagccg
     3421 atctcggctt gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact
     3481 tcttcccgta tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct
     3541 tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag attgatgagc
     3601 gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata gatatagatc
     3661 tcactacgcg gctgctcaaa cttgggcaga acgtaagccg cgagagcgcc aacaaccgct
     3721 tcttggtcga aggcagcaag cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa
     3781 tcggagtccg gctgatgttg ggagtaggtg gctacgtctc cgaactcacg accgaaaaga
     3841 tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt gcgaatgatg
     3901 cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt aacatcgttg
     3961 ctgctccata acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga
     4021 ggcatagact gtacaaaaaa acagtcataa caagccatga aaaccgccac tgcgccgtta
     4081 ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg agcgcatacg ctacttgcat
     4141 tacagtttac gaaccgaaca ggcttatgtc aactgggttc gtgccttcat ccgtttccac
     4201 ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg aggcatttct gtcctggctg
     4261 gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg cattggcggc cttgctgttc
     4321 ttctacggca aggtgctgtg cacggatcta ccatgggatc ctcgatcaac tttgtataga
     4381 aaagttgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
     4441 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gtcgacctgc
     4501 agactggctg tgtataaggg agcctgacat ttatattccc cagaacatca ggttaatggc
     4561 gtttttgatg tcattttcgc ggtggctgag atcagccact tcttccccga taacggagac
     4621 cggcacactg gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac
     4681 cgggtaaagt tcacggggga ctttatctga cagcagacgt gcactggcca gggggatcac
     4741 catccgtcgc ccgggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg
     4801 gctctctctt ttataggtgt aaaccttaaa ctgcatttca ccagcccctg ttctcgtcgg
     4861 caaaagagcc gttcatttca ataaaccggg cgacctcagc catcccttcc tgattttccg
     4921 ctttccagcg ttcggcacgc agacgacggg cttcattctg catggttgtg cttaccgaac
     4981 cggagatatt gacatcatat atgccttgag caactgatag ctgtcgctgt caactgtcac
     5041 tgtaatacgc tgcttcatag catacctctt tttgacatac ttcgggtata catatcagta
     5101 tatattctta taccgcaaaa atcagcgcgc aaatacgcat actgttatct ggcttttagt
     5161 aagccggatc ctctagatta cgccccgcct gccactcatc gcagtactgt tgtaattcat
     5221 taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
     5281 gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga
     5341 agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg
     5401 agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac
     5461 acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc
     5521 agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat
     5581 cccatatcac cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca
     5641 ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct
     5701 ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact
     5761 gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag
     5821 tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgacgga tcctaactca
     5881 aaatccacac attatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgcgg
     5941 ccgccatagt gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
     6001 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa ctttattata
     6061 catagttgat cgagctagca
//
