LOCUS       Exported                6923 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synchronize trafficking of ManII from the ER (RUSH system).
ACCESSION   .
VERSION     .
KEYWORDS    Ii-Str_ManII-SBP-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6923)
  AUTHORS   Boncompain G, Divoux S, Gareil N, de Forges H, Lescure A, Latreche 
            L, Mercanti V, Jollivet F, Raposo G, Perez F
  TITLE     Synchronization of secretory protein traffic in populations of 
            cells.
  JOURNAL   Nat Methods. 2012 Mar 11;9(5):493-8. doi: 10.1038/nmeth.1928.
  PUBMED    22406856
REFERENCE   2  (bases 1 to 6923)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6923
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(48..67)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        239..618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..822
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     773..793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     867..886
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        867..885
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1753..1779
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     intron          2298..2527
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2510..2529
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2585..3158
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2752..2769)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2979..2998
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3560..3670
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             3680..4393
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             3680..4393
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3722..3743)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3983..4002)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4330..4351
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4547..4668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4584..4603)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4770..4786)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4770..4786)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4783..4805)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4794..4810
                     /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(4818..4848)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4863..4884
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5001..5018)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5172..5760)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5252..5271)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5931..6791)
                     /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     6554..6573
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6792..6896)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgtcgacgga tcgggagatc tcccgatccc ctatggtcga ctctcagtac aatctgctct
       61 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      181 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      241 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      301 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      361 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      421 ttccattgac gtcaatgggt ggactattta cggtaaactg cccacttggc agtacatcaa
      481 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa
      841 cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagcttgg
      901 taccgagctc ggatcgatat ctgcggccta gctagcgctt aaggcctgtt aaccggtcgt
      961 acgtctccgg attcgaatta attcggatcc gtgatgcaca ggaggagagc cagggcctgt
     1021 cgggaagatc aaaagccagt catcgatgat cagcgcgacc ttatctccaa caatgagcaa
     1081 ctgcccatgc tgggccggcg ccctggggcc ccggagagca agtgcagccg cggagccctg
     1141 tacacaggct tttccatcct ggtgactctg ctcctcgctg gccaggccac caccgcctac
     1201 ttcctgtacc agcagcaggg ccggctggac aaactgacag tcacctccca gaacctgcag
     1261 ctcgagatgg accctagcaa agactcaaaa gctcaggtgt ccgctgccga ggctggcatt
     1321 actggaacat ggtacaatca gctcgggagc acctttattg tgactgctgg agccgatgga
     1381 gccctcaccg gaacatacga atctgctgtg ggaaacgccg aatcacggta cgtcctcact
     1441 ggccgatacg atagtgcccc tgccaccgac ggatctggga ctgccctggg atggactgtc
     1501 gcttggaaaa acaactaccg gaatgctcat tctgccacaa catggagtgg acagtacgtg
     1561 ggaggcgctg aggctagaat caatacacag tggctgctca catctggcac aaccgaggca
     1621 aatgcttgga aatccaccct ggtgggacat gacacattca ccaaagtgaa accctccgcc
     1681 gcttcaatcg atgccgccaa aaaagccgga gtcaacaacg gcaatcctct ggatgccgtc
     1741 cagcaggtcg actatccgta cgacgtacca gactacgcac tcgacctgca gctggagaac
     1801 ctgcgcatga agcttcccaa gcctcccaag cctgtgagca agatgcgcat ggccaccccg
     1861 ctgctgatgc atgcgctgcc catgggagcc ctgccccagg ggcccatgca gaatgccacc
     1921 aagtatggca acatgacaga ggaccatgtg atgcacctgc tccagaatgc tgaccccctg
     1981 aaggtgtacc cgccactgaa ggggagcttc ccggagaacc tgagacacct taagaacacc
     2041 atggagacca tagactggaa ggtctttgag agctggatgc accattggct cctgtttgaa
     2101 atgagcaggc actccttgga gcaaaagccc actgacgctc caccgaaaga gtcactggaa
     2161 ctggaggacc cgtcttctgg gctgggtgtg accaagcagg atctgggccc agtccccatg
     2221 tgaggatccg cggccgcata gataactgat ccagtgtgct ggaattaatt cgctgtctgc
     2281 gagggccagc tgttggggtg agtactccct ctcaaaagcg ggcatgactt ctgcgctaag
     2341 attgtcagtt tccaaaaacg aggaggattt gatattcacc tggcccgcgg tgatgccttt
     2401 gagggtggcc gcgtccatct ggtcagaaaa gacaatcttt ttgttgtcaa gcttgaggtg
     2461 tggcaggctt gagatctggc catacacttg agtgacaatg acatccactt tgcctttctc
     2521 tccacaggtg tccactccca ggtccaactg caggtcgagc atgcatctag ggcggccaat
     2581 tccgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     2641 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     2701 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     2761 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     2821 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     2881 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     2941 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     3001 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     3061 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     3121 acggggacgt ggttttcctt tgaaaaacac gatgataagc ttgccacaac ccgggaggcg
     3181 cgccatgaag ttaagtcgcc agttcaccgt gtttggcagc gcgatcttct gcgtcgtaat
     3241 cttctcactc tacctgatgc tggacagggg tcacttggac taccctcggg gcccgcgcca
     3301 ggagggctcc tttccgcagg gccagctttc aatattgcaa gaaaagattg accatttgga
     3361 gcgtttgctc gctgagaaca acgagattat ctcaaatatc agagactcag tcatcaacct
     3421 gagcgagtct gtggaggacg gcccgcgggg gtcaccaggc aacgccagcc aaggctccat
     3481 ccacctccac tcgccacagt tggccctgca ggctgacccc agagactgtt tggatcccac
     3541 cggtcgccac cggaattccg acgagaagac cactggttgg cgaggtggac acgttgttga
     3601 aggactggct ggggaacttg aacaacttcg tgcacgactg gagcatcacc cacaaggtca
     3661 acgtgaacca cctgcaggtg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     3721 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     3781 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     3841 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     3901 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     3961 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     4021 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     4081 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     4141 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     4201 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     4261 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     4321 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     4381 cgagctgtac aagggccggc cttaaggcct cgagggccaa ttaattaact ctagataact
     4441 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     4501 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     4561 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4621 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtcgagtg
     4681 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4741 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4801 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4861 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4921 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4981 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5041 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5101 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5161 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5221 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5281 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5341 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     5401 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5461 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5521 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5581 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     5641 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5701 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5761 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     5821 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     5881 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5941 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6001 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6061 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6121 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6181 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6241 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6301 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6361 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6421 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6481 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6541 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6601 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6661 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6721 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6781 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     6841 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     6901 tttccccgaa aagtgccacc tga
//
