LOCUS       sequence-328221-        6658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RUSH cargo: Str-KDEL_IRES_Tumor Necrosis Factor alpha 
            (TNFa)-SBP-mApple.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6658)
  AUTHORS   Weigel AV, Chang CL, Shtengel G, Xu CS, Hoffman DP, Freeman M, Iyer 
            N, Aaron J, Khuon S, Bogovic J, Qiu W, Hess HF, Lippincott-Schwartz 
            J
  TITLE     ER-to-Golgi protein delivery through an interwoven, tubular network 
            extending from ER.
  JOURNAL   Cell. 2021 Apr 29;184(9):2412-2429.e16. doi: 
            10.1016/j.cell.2021.03.035. Epub 2021 Apr 13.
  PUBMED    33852913
REFERENCE   2  (bases 1 to 6658)
  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: 
            "10.1016/j.cell.2021.03.035"; journalName: "Cell"; date: 
            "2021-04-29- 29"; volume: "184"; issue: "9"; pages: "2412-2429.e16"
COMMENT     Sequence Label: mApple-TNFa-RUSH
FEATURES             Location/Qualifiers
     source          1..6658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        104..483
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        484..687
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     638..658
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     732..751
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        732..750
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1515..1744
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1727..1746
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    1816..2389
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1983..2000)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2210..2229
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3160..3273
                     /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             3283..3990
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNF
                     EDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALK
                     SEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     3696..3715
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4147..4268
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4184..4203)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4370..4386)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4370..4386)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4383..4405)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4394..4410
                     /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(4418..4424,4425..4442,4443..4448))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4463..4484
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4601..4618)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4772..5360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4852..4871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5531..6322,6323..6391))
                     /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     6154..6173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6392..6496)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6571..6590)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
       61 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
      121 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      181 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      241 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      301 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      361 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      421 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      481 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      541 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      601 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      661 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
      721 cttatcgaaa ttaatacgac tcactatagg gagacccaag cttggtaccg agctcggatc
      781 gatatctgcg gcctagctag cgcttaaggc ctgttaaccg gtatggatgt atgcgtccgt
      841 cttgccctgt ggctcctctg gggactcctc ctgcaccagg gccagagcct cagccatagt
      901 cacagtgaga aggcgacagg aaccagctcg ggcgccggtt taaacgaccc tagcaaagac
      961 tcaaaagctc aggtgtccgc tgccgaggct ggcattactg gaacatggta caatcagctc
     1021 gggagcacct ttattgtgac tgctggagcc gatggagccc tcaccggaac atacgaatct
     1081 gctgtgggaa acgccgaatc acggtacgtc ctcactggcc gatacgatag tgcccctgcc
     1141 accgacggat ctgggactgc cctgggatgg actgtcgctt ggaaaaacaa ctaccggaat
     1201 gctcattctg ccacaacatg gagtggacag tacgtgggag gcgctgaggc tagaatcaat
     1261 acacagtggc tgctcacatc tggcacaacc gaggcaaatg cttggaaatc caccctggtg
     1321 ggacatgaca cattcaccaa agtgaaaccc tccgccgctt caatcgatgc cgccaaaaaa
     1381 gccggagtca acaacggcaa tcctctggat gccgtccagc agctcggacc gaaagatgaa
     1441 ctgtgagcgg ccgcatagat aactgatcca gtgtgctgga attaattcgc tgtctgcgag
     1501 ggccagctgt tggggtgagt actccctctc aaaagcgggc atgacttctg cgctaagatt
     1561 gtcagtttcc aaaaacgagg aggatttgat attcacctgg cccgcggtga tgcctttgag
     1621 ggtggccgcg tccatctggt cagaaaagac aatctttttg ttgtcaagct tgaggtgtgg
     1681 caggcttgag atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc
     1741 acaggtgtcc actcccaggt ccaactgcag gtcgagcatg catctagggc ggccaattcc
     1801 gcccctctcc ccccccccnt ctccctcccc cccccctaac gttactggcc gaagccgctt
     1861 ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg
     1921 caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc
     1981 ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga
     2041 agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc
     2101 tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc
     2161 acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc
     2221 aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga
     2281 tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg
     2341 ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataag cttgccacaa
     2401 cccgggaggc gcgccatgtc taccgagtct atgattaggg acgtggaact ggctgaggag
     2461 gcactgccca aaaaaaccgg cggaccacag ggctctagga gatgtctgtt tctgtctctg
     2521 ttctcttttc tcatcgtggc tggcgctaca acactcttct gtctgctcca tttcggcgtg
     2581 attggaccac agcgagagga atttccccgg gatctgtcac tcatttcacc actggcacag
     2641 gctgtccgat cttcatctcg gactccatcc gacaaacctg tcgcccatgt cgtcgccaac
     2701 ccacaggccg agggccagct ccagtggctc aataggaggg caaacgctct gctcgccaat
     2761 ggcgtggaac tccgggataa ccagctcgtc gtgcctagtg agggactgta cctcatctac
     2821 tcccaggtgc tgtttaaggg ccagggatgt ccttctacac atgtgctgct cacacacaca
     2881 atttcacgga tcgccgtgtc ttaccagact aaagtcaatc tgctctctgc catcaaatcc
     2941 ccatgtcagc gggaaacacc tgagggcgct gaggctaaac cttggtacga acccatctac
     3001 ctcggaggcg tgttccagct ggagaagggc gatagactga gtgctgagat caatcgaccc
     3061 gactacctcg actttgccga atctggccag gtctactttg gcatcattgc tctgggcctc
     3121 gagggccgag ctcatggcgc acctaggcct ttgaattcca tggacgagaa gaccactggt
     3181 tggcgaggtg gacacgttgt tgaaggactg gctggggaac ttgaacaact tcgtgcacga
     3241 ctggagcatc acccacaagg tcaacgtgaa ccacctgcag gtatggtgag caagggcgag
     3301 gagaataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     3361 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgaggccttt
     3421 cagaccgcta agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     3481 tcccctcagt tcatgtacgg ctccaaggtc tacattaagc acccagccga catccccgac
     3541 tacttcaagc tgtccttccc cgagggcttc aggtgggagc gcgtgatgaa cttcgaggac
     3601 ggcggcatta ttcacgttaa ccaggactcc tccctgcaag acggcgtgtt catctacaag
     3661 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     3721 ggctgggagg cctccgagga gcggatgtac cccgaggacg gcgccctgaa gagcgagatc
     3781 aagaagaggc tgaagctgaa ggacggcggc cactacgccg ccgaggtcaa gaccacctac
     3841 aaggccaaga agcccgtgca gctgcccggc gcctacatcg tcgacatcaa gttggacatc
     3901 gtgtcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     3961 tccaccggcg gcatggacga gctgtacaag tccggccggc cttaaggcct cgagggccaa
     4021 ttaattaact ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     4081 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     4141 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     4201 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     4261 gtatcttaac gcgtcgagtg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4321 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4381 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4441 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4501 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4561 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4621 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4681 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4741 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4801 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4861 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4921 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4981 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5041 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5101 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5161 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5221 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5281 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5341 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5401 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5461 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5521 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5581 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5641 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5701 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5761 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5821 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5881 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5941 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6001 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6061 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6121 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6181 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6241 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6301 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6361 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6421 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6481 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     6541 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     6601 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaatt
//
