LOCUS       sequence-120696-        6390 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 6390)
  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 6390)
  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-EGFP_puromycin
FEATURES             Location/Qualifiers
     source          1..6390
                     /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             join(1443..1445,1446..1448,1449..2159)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1443..1445,1446..1448,1449..2159)
                     /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(1488..1509)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1749..1768)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2096..2117
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     intron          2269..2498
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2481..2500
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2571..3145,3146..3148,3149..3157)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2738..2755)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2965..2984
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3177..3776
                     /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(3177..3196)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3673..3693
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4010..4131
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4047..4066)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4233..4249)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4233..4249)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4246..4268)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4257..4273
                     /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(4281..4287,4288..4305,4306..4311))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4326..4347
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4464..4481)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4635..5223)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4715..4734)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5394..6185,6186..6254))
                     /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     6017..6036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6255..6359)
                     /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 caaaggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1501 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1561 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1621 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1681 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1741 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1801 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1861 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1921 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1981 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2041 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     2101 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     2161 gccggcctta aggcctcgag ggccaattaa ttaaggatcc gcggccgcat agataactga
     2221 tccagtgtgc tggaattaat tcgctgtctg cgagggccag ctgttggggt gagtactccc
     2281 tctcaaaagc gggcatgact tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt
     2341 tgatattcac ctggcccgcg gtgatgcctt tgagggtggc cgcgtccatc tggtcagaaa
     2401 agacaatctt tttgttgtca agcttgaggt gtggcaggct tgagatctgg ccatacactt
     2461 gagtgacaat gacatccact ttgcctttct ctccacaggt gtccactccc aggtccaact
     2521 gcaggtcgag catgcatcta gggcggccaa ttccgcccct ctcccccccc cccctctccc
     2581 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     2641 tatatgtgat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     2701 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     2761 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     2821 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     2881 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     2941 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     3001 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     3061 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     3121 ttcctttgaa aaacacgatg ataagcttgc cacaacccac aaggagacga ccttccatga
     3181 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca
     3241 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgac ccggaccgcc
     3301 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     3361 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     3421 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     3481 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     3541 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     3601 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     3661 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     3721 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc
     3781 cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatggc tccgaccgaa
     3841 gccacccggg gcggccccgc cgaccccgca cccgcccccg aggcccaccg actctagata
     3901 actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     3961 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     4021 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4081 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtcga
     4141 gtgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4201 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4261 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4321 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4381 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4441 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4501 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4561 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4621 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4681 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4741 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4801 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     4861 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4921 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4981 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5041 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     5101 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5161 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5221 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5281 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5341 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5401 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5461 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5521 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5581 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5641 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5701 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5761 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5821 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5881 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5941 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6001 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6061 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6121 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6181 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6241 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6301 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6361 acatttcccc gaaaagtgcc acctgacgtc
//
