LOCUS       sequence-335802-        5933 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses ER-targeted streptavidin-KDEL (hook) and SBP-DsRed2 
            targeted to the ER lumen with an N-terminal EPV tripeptide (cargo) 
            for RUSH..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5933)
  AUTHORS   Yan R, Chen K, Wang B, Xu K
  TITLE     SURF4-induced tubular ERGIC selectively expedites ER-to-Golgi 
            transport.
  JOURNAL   Dev Cell. 2022 Feb 28;57(4):512-525.e8. doi: 
            10.1016/j.devcel.2021.12.018. Epub 2022 Jan 19.
  PUBMED    35051356
REFERENCE   2  (bases 1 to 5933)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.devcel.2021.12.018"; journalName: "Dev Cell"; date: 
            "2022-02-28- 28"; volume: "57"; issue: "4"; pages: "512-525.e8"
COMMENT     Sequence Label: pStr-KDEL_EPV-SBP-DsRed2-ER-5
FEATURES             Location/Qualifiers
     source          1..5933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1412..1641
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1624..1643
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1713..2287,2288..2290,2291..2299)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1880..1897)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2107..2126
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2379..2489
                     /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"
     regulatory      2499..2508
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2505..3179
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(2685..2705)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     3097..3120
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3319..3440
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3356..3375)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3542..3558)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3542..3558)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3555..3577)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3566..3582
                     /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(3590..3596,3597..3614,3615..3620))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3635..3656
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3773..3790)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3944..4532)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4024..4043)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4703..5494,5495..5563))
                     /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     5326..5345
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5564..5668)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5743..5762)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatcgat atctgcggcc tagctagcgc ttaaggcctg ttaaccggta
      721 tggatgtatg cgtccgtctt gccctgtggc tcctctgggg actcctcctg caccagggcc
      781 agagcctcag ccatagtcac agtgagaagg cgacaggaac cagctcgggc gccggtttaa
      841 acgaccctag caaagactca aaagctcagg tgtccgctgc cgaggctggc attactggaa
      901 catggtacaa tcagctcggg agcaccttta ttgtgactgc tggagccgat ggagccctca
      961 ccggaacata cgaatctgct gtgggaaacg ccgaatcacg gtacgtcctc actggccgat
     1021 acgatagtgc ccctgccacc gacggatctg ggactgccct gggatggact gtcgcttgga
     1081 aaaacaacta ccggaatgct cattctgcca caacatggag tggacagtac gtgggaggcg
     1141 ctgaggctag aatcaataca cagtggctgc tcacatctgg cacaaccgag gcaaatgctt
     1201 ggaaatccac cctggtggga catgacacat tcaccaaagt gaaaccctcc gccgcttcaa
     1261 tcgatgccgc caaaaaagcc ggagtcaaca acggcaatcc tctggatgcc gtccagcagc
     1321 tcggaccgaa agatgaactg tgagcggccg catagataac tgatccagtg tgctggaatt
     1381 aattcgctgt ctgcgagggc cagctgttgg ggtgagtact ccctctcaaa agcgggcatg
     1441 acttctgcgc taagattgtc agtttccaaa aacgaggagg atttgatatt cacctggccc
     1501 gcggtgatgc ctttgagggt ggccgcgtcc atctggtcag aaaagacaat ctttttgttg
     1561 tcaagcttga ggtgtggcag gcttgagatc tggccataca cttgagtgac aatgacatcc
     1621 actttgcctt tctctccaca ggtgtccact cccaggtcca actgcaggtc gagcatgcat
     1681 ctagggcggc caattccgcc cctctccccc cccccctctc cctccccccc ccctaacgtt
     1741 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
     1801 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
     1861 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag
     1921 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
     1981 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat
     2041 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
     2101 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
     2161 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg
     2221 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
     2281 tgataagctt gccacaaccc gggaggcgcg ccatgctgct atccgtgccg ttgctgctcg
     2341 gcctcctcgg cctggccgtc gccgaaccgg tcgccaccga cgagaagacc actggttggc
     2401 gaggtggaca cgttgttgaa ggactggctg gggaacttga acaacttcgt gcacgactgg
     2461 agcatcaccc acaaggtcaa cgtgaaccag caccggtcgc caccatggcc tcctccgaga
     2521 acgtcatcac cgagttcatg cgcttcaagg tgcgcatgga gggcaccgtg aacggccacg
     2581 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggccacaac accgtgaagc
     2641 tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttcc
     2701 agtacggctc caaggtgtac gtgaagcacc ccgccgacat ccccgactac aagaagctgt
     2761 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggcga
     2821 ccgtgaccca ggactcctcc ctgcaggacg gctgcttcat ctacaaggtg aagttcatcg
     2881 gcgtgaactt cccctccgac ggccccgtga tgcagaagaa gaccatgggc tgggaggcct
     2941 ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg cgagacccac aaggccctga
     3001 agctgaagga cggcggccac tacctggtgg agttcaagtc catctacatg gccaagaagc
     3061 ccgtgcagct gcccggctac tactacgtgg acgccaagct ggacatcacc tcccacaacg
     3121 aggactacac catcgtggag cagtacgagc gcaccgaggg ccgccaccac ctgttcctgt
     3181 ccggaaagga cgagctgtaa gtctagataa ctgatcataa tcagccatac cacatttgta
     3241 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     3301 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3361 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3421 aaactcatca atgtatctta acgcgtcgag tgcattctag ttgtggtttg tccaaactca
     3481 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     3541 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3601 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3661 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3721 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3781 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3841 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3901 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3961 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4021 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4081 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4141 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4201 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4261 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4321 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4381 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4441 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4501 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4561 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4621 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4681 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4741 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4801 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4861 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4921 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4981 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5041 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5101 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5161 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5221 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5281 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5341 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5401 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5461 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5521 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5581 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5641 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     5701 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     5761 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     5821 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     5881 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
