LOCUS       sequence-327170-        5989 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite Gateway (attR4-attR3) vector for mammalian protein 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5989)
  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 5989)
  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-312 (attR4-attR3)
FEATURES             Location/Qualifiers
     source          1..5989
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    407..530
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(571..876)
                     /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(673..692)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1196..1876)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     1791..1810
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1930..1936,1937..1954,1955..1960))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(1985..2108)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2155..2171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2155..2171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2168..2190)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2179..2195
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     promoter        complement(join(2203..2209,2210..2227,2228..2233))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2248..2269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2386..2403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2504..2521)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2510..2734
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2809..2832)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3175..3194
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3254..3853
                     /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(3254..3273)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3750..3770
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     rep_origin      complement(4170..4758)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4250..4269)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4929..5720,5721..5789))
                     /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     5552..5571
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5790..5894)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5962..5980
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagatcaac tttgtataga
      421 aaagttgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
      481 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gtcgacctgc
      541 agactggctg tgtataaggg agcctgacat ttatattccc cagaacatca ggttaatggc
      601 gtttttgatg tcattttcgc ggtggctgag atcagccact tcttccccga taacggagac
      661 cggcacactg gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac
      721 cgggtaaagt tcacggggga ctttatctga cagcagacgt gcactggcca gggggatcac
      781 catccgtcgc ccgggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg
      841 gctctctctt ttataggtgt aaaccttaaa ctgcatttca ccagcccctg ttctcgtcgg
      901 caaaagagcc gttcatttca ataaaccggg cgacctcagc catcccttcc tgattttccg
      961 ctttccagcg ttcggcacgc agacgacggg cttcattctg catggttgtg cttaccgaac
     1021 cggagatatt gacatcatat atgccttgag caactgatag ctgtcgctgt caactgtcac
     1081 tgtaatacgc tgcttcatag catacctctt tttgacatac ttcgggtata catatcagta
     1141 tatattctta taccgcaaaa atcagcgcgc aaatacgcat actgttatct ggcttttagt
     1201 aagccggatc ctctagatta cgccccgcct gccactcatc gcagtactgt tgtaattcat
     1261 taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
     1321 gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga
     1381 agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg
     1441 agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac
     1501 acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc
     1561 agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat
     1621 cccatatcac cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca
     1681 ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct
     1741 ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact
     1801 gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag
     1861 tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgacgga tcctaactca
     1921 aaatccacac attatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgcgg
     1981 ccgccatagt gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
     2041 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa ctttattata
     2101 catagttgat cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata
     2161 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2221 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2281 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2341 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2401 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2461 gttatccaca gaatcagggg ataacgcagg aaagaacatg tagcctcgac tgtgccttct
     2521 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2581 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2641 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2701 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2761 ggaagcttct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
     2821 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
     2881 tccaccggcc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc
     2941 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg
     3001 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg
     3061 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc
     3121 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
     3181 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt
     3241 cgaccagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     3301 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     3361 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     3421 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     3481 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     3541 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     3601 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     3661 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     3721 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     3781 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     3841 gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc
     3901 ccatgcatcg tagaattccg gacttgactc atgcttgttt cactttcaca tggaatttcc
     3961 cagttatgaa attaataaaa atcaatggtt tccacatctc tcagtgcctc tatctggagg
     4021 ccaggtaggg ctggccttgg gggaggggga ggccagaatg actccaagag ctacaggaag
     4081 gcaggtcaga gaccccaagc ttgaattcac atgtgagcaa aaggccagca aaaggccagg
     4141 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4201 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4261 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4321 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4381 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4441 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4501 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4561 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4621 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4681 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4741 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4801 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4861 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4921 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4981 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5041 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5101 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5161 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5221 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5281 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5341 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5401 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5461 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5521 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5581 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5641 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5701 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5761 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5821 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5881 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     5941 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
