LOCUS       Exported                6750 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  TNF reporter only (no hook) (RUSH system).
ACCESSION   .
VERSION     .
KEYWORDS    TNF-SBP-mCherry_puromycin
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6750)
  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 6750)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6750
                     /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..2519
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1957..1975)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2225..2244
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     intron          2629..2858
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2841..2860
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2931..3504
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3098..3115)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3325..3344
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3537..4136
                     /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(3537..3556)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4033..4053
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4370..4491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4407..4426)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4593..4609)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4593..4609)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4606..4628)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4617..4633
                     /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(4641..4671)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4686..4707
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4824..4841)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4995..5583)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5075..5094)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5754..6614)
                     /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     6377..6396
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6615..6719)
                     /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 aggataacat ggccatcatc aaggagttca
     1861 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     1921 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     1981 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     2041 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     2101 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     2161 ccctacagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     2221 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     2281 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     2341 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     2401 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     2461 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagg
     2521 gccggcctta aggcctcgag ggccaattaa ttaaggatcc gcggccgcat agataactga
     2581 tccagtgtgc tggaattaat tcgctgtctg cgagggccag ctgttggggt gagtactccc
     2641 tctcaaaagc gggcatgact tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt
     2701 tgatattcac ctggcccgcg gtgatgcctt tgagggtggc cgcgtccatc tggtcagaaa
     2761 agacaatctt tttgttgtca agcttgaggt gtggcaggct tgagatctgg ccatacactt
     2821 gagtgacaat gacatccact ttgcctttct ctccacaggt gtccactccc aggtccaact
     2881 gcaggtcgag catgcatcta gggcggccaa ttccgcccct ctcccccccc cccctctccc
     2941 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     3001 tatatgtgat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     3061 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     3121 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     3181 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     3241 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     3301 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     3361 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     3421 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     3481 ttcctttgaa aaacacgatg ataagcttgc cacaacccac aaggagacga ccttccatga
     3541 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca
     3601 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc
     3661 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     3721 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     3781 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     3841 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     3901 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     3961 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     4021 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     4081 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc
     4141 cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatggc tccgaccgaa
     4201 gccacccggg gcggccccgc cgaccccgca cccgcccccg aggcccaccg actctagata
     4261 actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     4321 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     4381 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4441 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtcga
     4501 gtgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4561 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4621 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4681 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4741 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4801 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4861 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4921 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4981 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5041 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5101 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5161 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     5221 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5281 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5341 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5401 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     5461 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5521 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5581 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5641 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5701 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5761 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5821 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5881 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5941 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6001 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6061 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6121 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6181 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6241 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6301 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6361 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6421 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6481 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6541 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6601 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6661 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6721 acatttcccc gaaaagtgcc acctgacgtc
//
