LOCUS       Exported                6759 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  TNF reporter only (no hook) (RUSH system).
ACCESSION   .
VERSION     .
KEYWORDS    TNF-SBP-EGFP_puromycin
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6759)
  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 6759)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6759
                     /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"
     CDS             1689..1802
                     /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="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             1812..2528
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             1812..2528
                     /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(1857..1878)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2118..2137)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2465..2486
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          2638..2867
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2850..2869
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2940..3513
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3107..3124)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3334..3353
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3546..4145
                     /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(3546..3565)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4042..4062
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4379..4500
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4416..4435)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4602..4618)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4602..4618)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4615..4637)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4626..4642
                     /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(4650..4680)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4695..4716
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4833..4850)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5004..5592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5084..5103)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5763..6623)
                     /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     6386..6405
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6624..6728)
                     /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 cgccatgtct accgagtcta
      961 tgattaggga cgtggaactg gctgaggagg cactgcccaa aaaaaccggc ggaccacagg
     1021 gctctaggag atgtctgttt ctgtctctgt tctcttttct catcgtggct ggcgctacaa
     1081 cactcttctg tctgctccat ttcggcgtga ttggaccaca gcgagaggaa tttccccggg
     1141 atctgtcact catttcacca ctggcacagg ctgtccgatc ttcatctcgg actccatccg
     1201 acaaacctgt cgcccatgtc gtcgccaacc cacaggccga gggccagctc cagtggctca
     1261 ataggagggc aaacgctctg ctcgccaatg gcgtggaact ccgggataac cagctcgtcg
     1321 tgcctagtga gggactgtac ctcatctact cccaggtgct gtttaagggc cagggatgtc
     1381 cttctacaca tgtgctgctc acacacacaa tttcacggat cgccgtgtct taccagacta
     1441 aagtcaatct gctctctgcc atcaaatccc catgtcagcg ggaaacacct gagggcgctg
     1501 aggctaaacc ttggtacgaa cccatctacc tcggaggcgt gttccagctg gagaagggcg
     1561 atagactgag tgctgagatc aatcgacccg actacctcga ctttgccgaa tctggccagg
     1621 tctactttgg catcattgct ctgggcctcg agggccgagc tcatggcgca cctaggcctt
     1681 tgaattccat ggacgagaag accactggtt ggcgaggtgg acacgttgtt gaaggactgg
     1741 ctggggaact tgaacaactt cgtgcacgac tggagcatca cccacaaggt caacgtgaac
     1801 cacctgcagg tatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1861 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1921 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1981 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     2041 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     2101 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     2161 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2221 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     2281 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2341 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2401 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     2461 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2521 tgtacaaggg ccggccttaa ggcctcgagg gccaattaat taaggatccg cggccgcata
     2581 gataactgat ccagtgtgct ggaattaatt cgctgtctgc gagggccagc tgttggggtg
     2641 agtactccct ctcaaaagcg ggcatgactt ctgcgctaag attgtcagtt tccaaaaacg
     2701 aggaggattt gatattcacc tggcccgcgg tgatgccttt gagggtggcc gcgtccatct
     2761 ggtcagaaaa gacaatcttt ttgttgtcaa gcttgaggtg tggcaggctt gagatctggc
     2821 catacacttg agtgacaatg acatccactt tgcctttctc tccacaggtg tccactccca
     2881 ggtccaactg caggtcgagc atgcatctag ggcggccaat tccgcccctc tccccccccc
     2941 ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
     3001 gcgtttgtct atatgtgatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     3061 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     3121 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
     3181 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     3241 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     3301 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     3361 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     3421 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
     3481 gacgtggttt tcctttgaaa aacacgatga taagcttgcc acaacccaca aggagacgac
     3541 cttccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtcccccggg
     3601 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgacc
     3661 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     3721 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     3781 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     3841 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     3901 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     3961 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     4021 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     4081 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     4141 cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatggct
     4201 ccgaccgaag ccacccgggg cggccccgcc gaccccgcac ccgcccccga ggcccaccga
     4261 ctctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     4321 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     4381 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4441 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4501 acgcgtcgag tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4561 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4621 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4681 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4741 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4801 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4861 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4921 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4981 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5041 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5101 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5161 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc
     5221 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5281 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5341 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5401 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5461 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5521 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5581 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5641 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5701 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5761 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5821 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5881 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5941 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6001 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6061 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6121 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6181 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6241 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6301 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6361 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6421 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6481 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6541 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6601 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6661 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6721 gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
