LOCUS       sequence-110469-        6381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ManII reporter only (no hook) (RUSH system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6381)
  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 6381)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2012-03-11- 11"; volume: "9"; 
            issue: "5"; pages: "493-8"
COMMENT     Sequence Label: ManII-SBP-mCherry_puromycin
FEATURES             Location/Qualifiers
     source          1..6381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             1320..1433
                     /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             1443..2150
                     /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(1588..1606)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1856..1875
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     intron          2260..2489
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2472..2491
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2562..3136,3137..3139,3140..3148)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2729..2746)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2956..2975
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3168..3767
                     /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(3168..3187)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3664..3684
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4001..4122
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4038..4057)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4224..4240)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4224..4240)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4237..4259)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4248..4264
                     /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(join(4272..4278,4279..4296,4297..4302))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4317..4338
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4455..4472)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4626..5214)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4706..4725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5385..6176,6177..6245))
                     /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     6008..6027
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6246..6350)
                     /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 cgccatgaag ttaagtcgcc
      961 agttcaccgt gtttggcagc gcgatcttct gcgtcgtaat cttctcactc tacctgatgc
     1021 tggacagggg tcacttggac taccctcggg gcccgcgcca ggagggctcc tttccgcagg
     1081 gccagctttc aatattgcaa gaaaagattg accatttgga gcgtttgctc gctgagaaca
     1141 acgagattat ctcaaatatc agagactcag tcatcaacct gagcgagtct gtggaggacg
     1201 gcccgcgggg gtcaccaggc aacgccagcc aaggctccat ccacctccac tcgccacagt
     1261 tggccctgca ggctgacccc agagactgtt tggatcccac cggtcgccac cggaattcca
     1321 tggacgagaa gaccactggt tggcgaggtg gacacgttgt tgaaggactg gctggggaac
     1381 ttgaacaact tcgtgcacga ctggagcatc acccacaagg tcaacgtgaa ccacctgcag
     1441 gtatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca
     1501 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     1561 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     1621 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc
     1681 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     1741 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctacagg
     1801 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     1861 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     1921 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg
     1981 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg
     2041 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     2101 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag ggccggcctt
     2161 aaggcctcga gggccaatta attaaggatc cgcggccgca tagataactg atccagtgtg
     2221 ctggaattaa ttcgctgtct gcgagggcca gctgttgggg tgagtactcc ctctcaaaag
     2281 cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat ttgatattca
     2341 cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa aagacaatct
     2401 ttttgttgtc aagcttgagg tgtggcaggc ttgagatctg gccatacact tgagtgacaa
     2461 tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac tgcaggtcga
     2521 gcatgcatct agggcggcca attccgcccc tctccccccc ccccctctcc ctcccccccc
     2581 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtga
     2641 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     2701 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     2761 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     2821 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     2881 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     2941 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     3001 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     3061 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     3121 aaaacacgat gataagcttg ccacaaccca caaggagacg accttccatg accgagtaca
     3181 agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg
     3241 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc cacatcgagc
     3301 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     3361 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     3421 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     3481 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     3541 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     3601 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     3661 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     3721 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg
     3781 acccgcagcg cccgaccgaa aggagcgcac gaccccatgg ctccgaccga agccacccgg
     3841 ggcggccccg ccgaccccgc acccgccccc gaggcccacc gactctagat aactgatcat
     3901 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     3961 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     4021 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4081 gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtcg agtgcattct
     4141 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4201 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4261 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4321 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4381 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4441 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4501 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4561 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4621 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4681 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4741 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4801 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4861 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4921 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4981 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5041 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     5101 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5161 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5221 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5281 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5341 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5401 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5461 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5521 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5581 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5641 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5701 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5761 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5821 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5881 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5941 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6001 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6061 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6121 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6181 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6241 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6301 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6361 cgaaaagtgc cacctgacgt c
//
