LOCUS       40924_10296        6925 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Encodes (N to C): GNSTM glycosylation motif, 3 residue spacer, Flag 
            tag,10 residue spacer, Lamp2b (exosomal transmembrane protein), 3 
            residue spacer, HA tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6925)
  AUTHORS   Hung ME, Leonard JN
  TITLE     Stabilization of exosome-targeting peptides via engineered 
            glycosylation.
  JOURNAL   J Biol Chem. 2015 Mar 27;290(13):8166-72. doi: 
            10.1074/jbc.M114.621383. Epub 2015 Feb 5.
  PUBMED    25657008
REFERENCE   2  (bases 1 to 6925)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6925)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1074/jbc.M114.621383"; journalName: "J Biol Chem"; date: 
            "2015-03-27- 27"; volume: "290"; issue: "13"; pages: "8166-72"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6925
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        490..693
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        738..756
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      779..788
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             893..916
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     CDS             2102..2128
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     polyA_signal    2231..2455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2501..2929
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2943..3272
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             3321..4343
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4476..4609
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4646..4662)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4670..4686)
                     /label=lac operator
                     /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(4694..4724)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4739..4760)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(4877..4894)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5048..5633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5807..6664)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6665..6769)
                     /label=AmpR promoter
     primer_bind     complement(6844..6863)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
ORIGIN
        1 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
       61 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      481 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      541 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      601 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      661 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      721 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagtgccgc
      781 cgccatggtg tgcttccgcc tcttcccggt tccgggctca gggctcgttc tggtctgcct
      841 agtcctggga gctgtgcggt cttatgcagg taactcgact atgggcagtg gagactataa
      901 agatgacgat gacaagggca gtggatctgg atccggtggc tcgagtttgg aacttaattt
      961 gacagattca gaaaatgcca cttgccttta tgcaaaatgg cagatgaatt tcacagttcg
     1021 ctatgaaact acaaataaaa cttataaaac tgtaaccatt tcagaccatg gcactgtgac
     1081 atataatgga agcatttgtg gggatgatca gaatggtccc aaaatagcag tgcagttcgg
     1141 acctggcttt tcctggattg cgaattttac caaggcagca tctacttatt caattgacag
     1201 cgtctcattt tcctacaaca ctggtgataa cacaacattt cctgatgctg aagataaagg
     1261 aattcttact gttgatgaac ttttggccat cagaattcca ttgaatgacc tttttagatg
     1321 caatagttta tcaactttgg aaaagaatga tgttgtccaa cactactggg atgttcttgt
     1381 acaagctttt gtccaaaatg gcacagtgag cacaaatgag ttcctgtgtg ataaagacaa
     1441 aacttcaaca gtggcaccca ccatacacac cactgtgcca tctcctacta caacacctac
     1501 tccaaaggaa aaaccagaag ctggaaccta ttcagttaat aatggcaatg atacttgcct
     1561 gctggctacc atggggctgc agctgaacat cactcaggat aaggttgctt cagttattaa
     1621 catcaacccc aatacaactc actccacagg cagctgccgt tctcacactg ctctacttag
     1681 actcaatagc agcactatta agtatctaga ctttgtcttt gctgtgaaaa atgaaaaccg
     1741 attttatctg aaggaagtga acatcagcat gtatttggtt aatggctccg ttttcagcat
     1801 tgcaaataac aatctcagct actgggatgc ccccctggga agttcttata tgtgcaacaa
     1861 agagcagact gtttcagtgt ctggagcatt tcagataaat acctttgatc taagggttca
     1921 gcctttcaat gtgacacaag gaaagtattc tacagcccaa gagtgttcgc tggatgatga
     1981 caccattcta atcccaatta tagttggtgc tggtctttca ggcttgatta tcgttatagt
     2041 gattgcttac gtaattggca gaagaaaaag ttatgctgga tatcagactc tgggaagcgg
     2101 atacccatac gatgtgccag attacgctta aggatccact agtccagtgt ggtggaattc
     2161 tgcagatatc cagcacagtg gcggccgctc gagtctagag ggcccgttta aacccgctga
     2221 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2281 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2341 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2401 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2461 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2521 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2581 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2641 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2701 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2761 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2821 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2881 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     2941 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3001 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     3061 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     3121 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3181 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3241 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3301 tcggatctga tcagcacgtg atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     3361 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     3421 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     3481 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     3541 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     3601 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     3661 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     3721 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     3781 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     3841 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     3901 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     3961 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     4021 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     4081 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     4141 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     4201 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     4261 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     4321 gcactcgtcc gagggcaaag gaatagcacg tgctacgaga tttcgattcc accgccgcct
     4381 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4441 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4501 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4561 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     4621 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4681 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4741 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4801 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4861 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4921 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4981 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5041 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5101 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5161 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5221 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5281 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5341 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5401 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5461 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5521 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5581 gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5641 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5701 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5761 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5821 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5881 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5941 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6001 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6061 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6121 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6181 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6241 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6301 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6361 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6421 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6481 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6541 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6601 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6661 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6721 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6781 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     6841 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     6901 ggaggtcgct gagtagtgcg cgagc
//
