LOCUS       sequence-322147-        4384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway (attR4-attR2) vector for mammalian protein 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4384)
  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 4384)
  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-302 (attR4-attR2)
FEATURES             Location/Qualifiers
     source          1..4384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     12..34
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     26..43
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     27..43
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    51..174
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(199..204,205..222,223..229)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             283..963
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(349..368)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1283..1588
                     /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     1467..1486
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1629..1752)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1772..1788)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1772..1788)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1785..1807)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(1881..1898)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1887..2111
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2213..2801)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2293..2312)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2972..3763,3764..3832))
                     /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     3595..3614
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3833..3937)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4005..4023
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4061..4083)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4183..4202
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattat caactttgta
       61 tagaaaagtt gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt
      121 gcataaaaaa cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc
      181 gcattaggca ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg
      241 agttaggatc cgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact
      301 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag
      361 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag
      421 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg
      481 atgaatgctc atccggaact ccgtatggca atgaaagacg gtgagctggt gatatgggat
      541 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg
      601 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt
      661 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca
      721 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc
      781 ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg
      841 ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat
      901 gaattacaac agtactgcga tgagtggcag gcggggcgta atctagagga tccggcttac
      961 taaaagccag ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata
     1021 ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc gtattacagt
     1081 gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa tatctccggt
     1141 ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg
     1201 gaaaatcagg aagggatggc tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct
     1261 gacgagaaca ggggctggtg aaatgcagtt taaggtttac acctataaaa gagagagccg
     1321 ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt
     1381 gatccccctg gccagtgcac gtctgctgtc agataaagtc ccccgtgaac tttacccggt
     1441 ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt
     1501 ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc
     1561 cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc
     1621 aggtcgacca tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat
     1681 gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt tcaactttct
     1741 tgtacaaagt tgatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     1801 atccgctggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1861 acgcaggaaa gaacatgtag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     1921 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     1981 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     2041 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     2101 gggctctatg gcttctgagg cggaaagaac cagctgggga agcttgaatt cacatgtgag
     2161 caaaaggcca gcaaaaggcc aagaaccgta aaaaggccgc gttgctggcg tttttccata
     2221 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2281 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2341 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2401 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2461 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2521 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2581 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2641 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2701 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2761 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2821 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2881 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2941 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3001 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3061 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3121 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3181 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3241 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3301 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3361 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3421 tcagaagtaa gttggctgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3481 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3541 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3601 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3661 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3721 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3781 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3841 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3901 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     3961 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     4021 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     4081 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     4141 tgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     4201 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     4261 gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     4321 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     4381 taac
//
