LOCUS       sequence-322148-        5718 bp    DNA     circular SYN 15-APR-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 5718)
  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 5718)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-305 (attR4-attR2)
FEATURES             Location/Qualifiers
     source          1..5718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..25
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     9..25
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    33..156
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(181..186,187..204,205..211)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             265..945
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(331..350)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1265..1570
                     /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     1449..1468
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1611..1734)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1754..1770)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1754..1770)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1767..1789)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     complement(1863..1880)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1869..2093
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2168..2191)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2534..2553
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2613..3212
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2613..2632)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3109..3129
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     rep_origin      complement(3529..4117)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3609..3628)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4288..5079,5080..5148))
                     /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     4911..4930
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5149..5253)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5321..5339
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5377..5399)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5499..5518
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(5712..5718,1..16)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgacgttgt aaaacgacgg ccagtgaatt atcaactttg tatagaaaag ttgaacgaga
       61 aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac
      121 ataatactgt aaaacacaac atatccagtc actatggcgg ccgcattagg caccccaggc
      181 tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga tccgtcgaga
      241 ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat
      301 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
      361 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
      421 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa
      481 ctccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac
      541 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
      601 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc
      661 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
      721 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc
      781 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
      841 catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
      901 gatgagtggc aggcggggcg taatctagag gatccggctt actaaaagcc agataacagt
      961 atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa
     1021 gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca gtgacagttg acagcgacag
     1081 ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg
     1141 cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg
     1201 gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa caggggctgg
     1261 tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
     1321 tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc
     1381 acgtctgctg tcagataaag tcccccgtga actttacccg gtggtgcata tcggggatga
     1441 aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga
     1501 agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg
     1561 gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgac catagtgact
     1621 ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata
     1681 tattgatatt tatatcattt tacgtttctc gttcaacttt cttgtacaaa gttgatagct
     1741 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctg gtatcagctc
     1801 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1861 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     1921 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     1981 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2041 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2101 ggcggaaaga accagctggg gaagcttcta ccgggtaggg gaggcgcttt tcccaaggca
     2161 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
     2221 gcctcgcaca cattccacat ccaccggccg gtaggcgcca accggctccg ttctttggtg
     2281 gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc
     2341 tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg
     2401 acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt
     2461 gctccttcgc tttctgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc
     2521 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
     2581 tcctcatctc cgggcctttc gaccagctta ccatgaccga gtacaagccc acggtgcgcc
     2641 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     2701 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     2761 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     2821 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     2881 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     2941 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     3001 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     3061 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     3121 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     3181 cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga
     3241 ccgaaaggag cgcacgaccc catgcatcgt agaattccgg acttgactca tgcttgtttc
     3301 actttcacat ggaatttccc agttatgaaa ttaataaaaa tcaatggttt ccacatctct
     3361 cagtgcctct atctggaggc caggtagggc tggccttggg ggagggggag gccagaatga
     3421 ctccaagagc tacaggaagg caggtcagag accccaagct tgaattcaca tgtgagcaaa
     3481 aggccagcaa aaggccaaga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3541 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3601 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3661 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3721 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3781 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3841 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3901 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3961 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4021 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4081 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4141 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4201 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4261 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4321 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4381 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4441 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4501 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4561 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4621 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4681 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4741 aagtaagttg gctgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4801 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4861 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4921 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4981 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5041 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5101 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5161 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5221 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5281 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     5341 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     5401 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgtgtc
     5461 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     5521 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     5581 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     5641 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     5701 gccagggttt tcccagtc
//
