LOCUS       Exported                8256 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Ecadherin reporter only (no hook) (RUSH system).
ACCESSION   .
VERSION     .
KEYWORDS    SBP-EGFP-Ecadherin_puromycin
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8256)
  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 8256)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8256
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     945..1004
                     /product="human interleukin-2 signal sequence"
                     /label=IL-2 signal sequence
     CDS             1005..1115
                     /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             1125..1841
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1125..1841
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1170..1191)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1431..1450)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1778..1799
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          4135..4364
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     4347..4366
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    4437..5010
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4604..4621)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4831..4850
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5043..5642
                     /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(5043..5062)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5539..5559
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5876..5997
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5913..5932)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(6099..6115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6099..6115)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6112..6134)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6123..6139
                     /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(6147..6177)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6192..6213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6330..6347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6501..7089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6581..6600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7260..8120)
                     /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     7883..7902
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8121..8225)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgatatctgc ggcctagcta gcgggaggcg cgccatgtac aggatgcaac
      961 tcctgtcttg cattgcacta agtcttgcac ttgtcacgaa ttccgacgag aagaccactg
     1021 gttggcgagg tggacacgtt gttgaaggac tggctgggga acttgaacaa cttcgtgcac
     1081 gactggagca tcacccacaa ggtcaacgtg aaccacctgc aggtatggtg agcaagggcg
     1141 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1201 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1261 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1321 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1381 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1441 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1501 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1561 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     1621 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1681 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     1741 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1801 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggccggcca gactgggtca
     1861 tccctcccat cagctgcccc gaaaatgaaa agggtgaatt cccaaagaac ctggttcaga
     1921 tcaaatccaa cagggacaaa gaaacaaagg ttttctacag catcaccggc caaggagctg
     1981 acaaaccccc cgttggcgtt ttcatcattg agagggagac aggctggctg aaagtgacac
     2041 agcctctgga tagagaagcc attgccaagt acatcctcta ttctcatgcc gtgtcatcaa
     2101 atggggaagc ggtggaggat cccatggaga tagtgatcac agtgacagat cagaatgaca
     2161 acaggccaga gtttacccag gaggtgtttg agggatccgt tgcagaaggc gctgttccag
     2221 gaacctccgt gatgaaggtc tcagccaccg atgcagacga tgacgtcaac acctacaacg
     2281 ctgccatcgc ctacaccatc gtcagccagg atcctgagct gcctcacaaa aacatgttca
     2341 ctgtcaatag ggacaccggg gtcatcagtg tgctcacctc tgggctggac cgagagagtt
     2401 accctacata cactctggtg gttcaggctg ctgaccttca aggcgaaggc ttgagcacaa
     2461 cagccaaggc tgtgatcact gtcaaggata ttaatgacaa cgctcctgtc ttcaacccga
     2521 gcacgtatca gggtcaagtg cctgagaatg aggtcaatgc ccggatcgcc acactcaaag
     2581 tgaccgatga tgatgccccc aacactccgg cgtggaaagc tgtgtacacc gtagtcaacg
     2641 atcctgacca gcagttcgtt gttgtcacag accccacgac caatgatggc attttgaaaa
     2701 cagccaaggg cttggatttt gaggccaagc agcaatacat ccttcatgtg agagtggaga
     2761 acgaggaacc ctttgagggg tctcttgtcc cttccacagc cactgtcact gtggacgtgg
     2821 tagacgtgaa tgaagccccc atctttatgc ctgcggagag gagagtcgaa gtgcccgaag
     2881 actttggtgt gggtcaggaa atcacatctt ataccgctcg agagccggac acgttcatgg
     2941 atcagaagat cacgtatcgg atttggaggg acactgccaa ctggctggag attaacccag
     3001 agactggtgc cattttcacg cgcgctgaga tggacagaga agacgctgag catgtgaaga
     3061 acagcacata tgtagctctc atcatcgcca cagatgatgg ttcacccatt gccactggca
     3121 cgggcactct tctcctggtc ctgttagacg tcaatgacaa cgctcccatc ccagaacctc
     3181 gaaacatgca gttctgccag aggaacccac agcctcatat catcaccatc ttggatccag
     3241 accttccccc caacacgtcc ccctttactg ctgagctaac ccatggggcc agcgtcaact
     3301 ggaccattga gtataatgac gcagctcaag aatctctcat tttgcaacca agaaaggact
     3361 tagagattgg cgaatacaaa atccatctca agctcgcgga taaccagaac aaagaccagg
     3421 tgaccacgtt ggacgtccat gtgtgtgact gtgaagggac ggtcaacaac tgcatgaagg
     3481 cgggaatcgt ggcagcagga ttgcaagttc ctgccatcct cggaatcctt ggagggatcc
     3541 tcgccctgct gattctgatc ctgctgctcc tactgtttct acggaggaga acggtggtca
     3601 aagagcccct gctgccacca gatgatgata cccgggacaa tgtgtattac tatgatgaag
     3661 aaggaggtgg agaagaagac caggactttg atttgagcca gctgcacagg ggcctggatg
     3721 cccgaccgga agtgactcga aatgatgtgg ctcccaccct catgagcgtg ccccagtatc
     3781 gtccccgtcc tgccaatcct gatgaaattg gaaacttcat cgatgaaaac ctgaaggcag
     3841 ccgacagcga ccccacggca cccccttacg actctctgtt ggtgttcgat tacgagggca
     3901 gtggttctga agccgctagc ctgagctcac tgaactcctc tgagtcggat caggaccagg
     3961 actacgatta tctgaacgag tggggcaacc gattcaagaa gctggcggac atgtacggcg
     4021 gtggcgaaga cgactagggc ctcgagggcc aattaattaa ggatccgcgg ccgcatagat
     4081 aactgatcca gtgtgctgga attaattcgc tgtctgcgag ggccagctgt tggggtgagt
     4141 actccctctc aaaagcgggc atgacttctg cgctaagatt gtcagtttcc aaaaacgagg
     4201 aggatttgat attcacctgg cccgcggtga tgcctttgag ggtggccgcg tccatctggt
     4261 cagaaaagac aatctttttg ttgtcaagct tgaggtgtgg caggcttgag atctggccat
     4321 acacttgagt gacaatgaca tccactttgc ctttctctcc acaggtgtcc actcccaggt
     4381 ccaactgcag gtcgagcatg catctagggc ggccaattcc gcccctctcc cccccccccc
     4441 tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg
     4501 tttgtctata tgtgattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa
     4561 cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg
     4621 caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca
     4681 acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc
     4741 ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt
     4801 gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg
     4861 ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca
     4921 tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac
     4981 gtggttttcc tttgaaaaac acgatgataa gcttgccaca acccacaagg agacgacctt
     5041 ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg
     5101 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg
     5161 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     5221 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     5281 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     5341 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     5401 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     5461 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     5521 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     5581 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     5641 gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catggctccg
     5701 accgaagcca cccggggcgg ccccgccgac cccgcacccg cccccgaggc ccaccgactc
     5761 tagataactg atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     5821 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     5881 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5941 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg
     6001 cgtcgagtgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     6061 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     6121 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     6181 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     6241 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6301 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     6361 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     6421 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6481 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6541 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6601 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6661 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
     6721 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6781 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6841 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6901 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     6961 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     7021 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     7081 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     7141 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     7201 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     7261 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     7321 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7381 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     7441 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     7501 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7561 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7621 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7681 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7741 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7801 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7861 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7921 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7981 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     8041 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     8101 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     8161 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     8221 ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
