LOCUS       40924_10291        6880 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Flag tag with 3 residue spacer fused to the N-terminus of Lamp2b, a 
            transmembrane exosomal protein. 3 residue spacer then HA tag fused 
            to the C-terminus of Lamp2b..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6880)
  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 6880)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6880)
  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..6880
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(160..745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(919..1776)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(1777..1881)
                     /label=AmpR promoter
     primer_bind     complement(1956..1975)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     enhancer        2147..2526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2527..2730
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        2775..2793
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2816..2825
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2906..2929
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     CDS             4094..4120
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     polyA_signal    4223..4447
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4493..4921
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4935..5264
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             5313..6335
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    6468..6601
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6638..6654)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(6662..6678)
                     /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(6686..6716)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6731..6752)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
ORIGIN
        1 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
       61 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
      121 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
      181 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
      241 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
      301 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
      361 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
      421 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
      481 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
      541 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
      601 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
      661 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt tttgtttgca agcagcagat
      721 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
      781 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
      841 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
      901 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
      961 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     1021 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     1081 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     1141 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     1201 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     1261 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     1321 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     1381 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     1441 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     1501 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     1561 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     1621 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     1681 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     1741 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     1801 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     1861 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     1921 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     1981 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     2041 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     2101 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
     2161 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     2221 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     2281 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     2341 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     2401 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     2461 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     2521 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     2581 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     2641 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     2701 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac
     2761 tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta gtgccgccgc
     2821 catggtgtgc ttccgcctct tcccggttcc gggctcaggg ctcgttctgg tctgcctagt
     2881 cctgggagct gtgcggtctt atgcagacta taaagatgac gatgacaagg gctcgagttt
     2941 ggaacttaat ttgacagatt cagaaaatgc cacttgcctt tatgcaaaat ggcagatgaa
     3001 tttcacagtt cgctatgaaa ctacaaataa aacttataaa actgtaacca tttcagacca
     3061 tggcactgtg acatataatg gaagcatttg tggggatgat cagaatggtc ccaaaatagc
     3121 agtgcagttc ggacctggct tttcctggat tgcgaatttt accaaggcag catctactta
     3181 ttcaattgac agcgtctcat tttcctacaa cactggtgat aacacaacat ttcctgatgc
     3241 tgaagataaa ggaattctta ctgttgatga acttttggcc atcagaattc cattgaatga
     3301 cctttttaga tgcaatagtt tatcaacttt ggaaaagaat gatgttgtcc aacactactg
     3361 ggatgttctt gtacaagctt ttgtccaaaa tggcacagtg agcacaaatg agttcctgtg
     3421 tgataaagac aaaacttcaa cagtggcacc caccatacac accactgtgc catctcctac
     3481 tacaacacct actccaaagg aaaaaccaga agctggaacc tattcagtta ataatggcaa
     3541 tgatacttgc ctgctggcta ccatggggct gcagctgaac atcactcagg ataaggttgc
     3601 ttcagttatt aacatcaacc ccaatacaac tcactccaca ggcagctgcc gttctcacac
     3661 tgctctactt agactcaata gcagcactat taagtatcta gactttgtct ttgctgtgaa
     3721 aaatgaaaac cgattttatc tgaaggaagt gaacatcagc atgtatttgg ttaatggctc
     3781 cgttttcagc attgcaaata acaatctcag ctactgggat gcccccctgg gaagttctta
     3841 tatgtgcaac aaagagcaga ctgtttcagt gtctggagca tttcagataa atacctttga
     3901 tctaagggtt cagcctttca atgtgacaca aggaaagtat tctacagccc aagagtgttc
     3961 gctggatgat gacaccattc taatcccaat tatagttggt gctggtcttt caggcttgat
     4021 tatcgttata gtgattgctt acgtaattgg cagaagaaaa agttatgctg gatatcagac
     4081 tctgggaagc ggatacccat acgatgtgcc agattacgct taaggatcca ctagtccagt
     4141 gtggtggaat tctgcagata tccagcacag tggcggccgc tcgagtctag agggcccgtt
     4201 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     4261 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4321 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4381 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     4441 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     4501 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4561 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4621 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4681 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     4741 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4801 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     4861 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4921 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     4981 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     5041 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     5101 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     5161 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     5221 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     5281 gtatatccat tttcggatct gatcagcacg tgatgaaaaa gcctgaactc accgcgacgt
     5341 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     5401 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     5461 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     5521 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     5581 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     5641 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     5701 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     5761 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     5821 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     5881 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     5941 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     6001 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     6061 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     6121 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     6181 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     6241 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     6301 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtgctacga gatttcgatt
     6361 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     6421 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     6481 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     6541 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     6601 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     6661 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     6721 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     6781 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6841 gtttgcgtat tgggcgctct tccgcttcct cgctcactga
//
