LOCUS       sequence-323914-        4376 bp    DNA     circular SYN 14-APR-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 4376)
  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 4376)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-311 (attR4-attR3)
FEATURES             Location/Qualifiers
     source          1..4376
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWVI"
     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"
     rep_origin      complement(2557..3145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2637..2656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3316..4107,4108..4176))
                     /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     3939..3958
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4177..4281)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4349..4367
                     /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 ttatattacc 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 tgagcaaaag gccagcaaaa
     2521 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2581 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2641 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2701 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2761 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2821 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2881 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2941 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3001 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3061 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3121 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3181 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3241 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3301 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3361 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3421 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3481 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3541 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3601 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3661 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3721 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3781 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3841 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3901 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3961 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4021 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4081 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4141 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4201 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4261 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     4321 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
