LOCUS       sequence-324053-        6821 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RUSH cargo: Str-KDEL_IRES_Tumor Necrosis Factor alpha 
            (TNFa)-SBP-Halo.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6821)
  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 6821)
  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: Halo-TNFa-RUSH
FEATURES             Location/Qualifiers
     source          1..6821
                     /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"
     intron          1646..1875
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1858..1877
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1933..2507,2508..2510,2511..2519)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2100..2117)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2327..2346
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3277..3390
                     /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             3406..4290
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     polyA_signal    4441..4562
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4478..4497)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4664..4680)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4664..4680)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4677..4699)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4688..4704
                     /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(4712..4718,4719..4736,4737..4742))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4757..4778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4895..4912)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5066..5654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5146..5165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5825..6616,6617..6685))
                     /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     6448..6467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6686..6790)
                     /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 gcgcttaagg cctgttaacc ggtatggatg
      961 tatgcgtccg tcttgccctg tggctcctct ggggactcct cctgcaccag ggccagagcc
     1021 tcagccatag tcacagtgag aaggcgacag gaaccagctc gggcgccggt ttaaacgacc
     1081 ctagcaaaga ctcaaaagct caggtgtccg ctgccgaggc tggcattact ggaacatggt
     1141 acaatcagct cgggagcacc tttattgtga ctgctggagc cgatggagcc ctcaccggaa
     1201 catacgaatc tgctgtggga aacgccgaat cacggtacgt cctcactggc cgatacgata
     1261 gtgcccctgc caccgacgga tctgggactg ccctgggatg gactgtcgct tggaaaaaca
     1321 actaccggaa tgctcattct gccacaacat ggagtggaca gtacgtggga ggcgctgagg
     1381 ctagaatcaa tacacagtgg ctgctcacat ctggcacaac cgaggcaaat gcttggaaat
     1441 ccaccctggt gggacatgac acattcacca aagtgaaacc ctccgccgct tcaatcgatg
     1501 ccgccaaaaa agccggagtc aacaacggca atcctctgga tgccgtccag cagctcggac
     1561 cgaaagatga actgtgagcg gccgcataga taactgatcc agtgtgctgg aattaattcg
     1621 ctgtctgcga gggccagctg ttggggtgag tactccctct caaaagcggg catgacttct
     1681 gcgctaagat tgtcagtttc caaaaacgag gaggatttga tattcacctg gcccgcggtg
     1741 atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt gttgtcaagc
     1801 ttgaggtgtg gcaggcttga gatctggcca tacacttgag tgacaatgac atccactttg
     1861 cctttctctc cacaggtgtc cactcccagg tccaactgca ggtcgagcat gcatctaggg
     1921 cggccaattc cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga
     1981 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     2041 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     2101 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     2161 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     2221 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     2281 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     2341 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     2401 ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc
     2461 cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataagctt gccacaaccc
     2521 gggaggcgcg ccatgtctac cgagtctatg attagggacg tggaactggc tgaggaggca
     2581 ctgcccaaaa aaaccggcgg accacagggc tctaggagat gtctgtttct gtctctgttc
     2641 tcttttctca tcgtggctgg cgctacaaca ctcttctgtc tgctccattt cggcgtgatt
     2701 ggaccacagc gagaggaatt tccccgggat ctgtcactca tttcaccact ggcacaggct
     2761 gtccgatctt catctcggac tccatccgac aaacctgtcg cccatgtcgt cgccaaccca
     2821 caggccgagg gccagctcca gtggctcaat aggagggcaa acgctctgct cgccaatggc
     2881 gtggaactcc gggataacca gctcgtcgtg cctagtgagg gactgtacct catctactcc
     2941 caggtgctgt ttaagggcca gggatgtcct tctacacatg tgctgctcac acacacaatt
     3001 tcacggatcg ccgtgtctta ccagactaaa gtcaatctgc tctctgccat caaatcccca
     3061 tgtcagcggg aaacacctga gggcgctgag gctaaacctt ggtacgaacc catctacctc
     3121 ggaggcgtgt tccagctgga gaagggcgat agactgagtg ctgagatcaa tcgacccgac
     3181 tacctcgact ttgccgaatc tggccaggtc tactttggca tcattgctct gggcctcgag
     3241 ggccgagctc atggcgcacc taggcctttg aattccatgg acgagaagac cactggttgg
     3301 cgaggtggac acgttgttga aggactggct ggggaacttg aacaacttcg tgcacgactg
     3361 gagcatcacc cacaaggtca acgtgaacca cctgcaggtg gatccgaaat cggtactggc
     3421 tttccattcg acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt
     3481 ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg
     3541 tggcgcaaca tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc
     3601 ggtatgggca aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc
     3661 atggatgcct tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg
     3721 ggctccgctc tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca
     3781 tttatggagt tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag
     3841 accttccagg ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt
     3901 tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac
     3961 cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac
     4021 gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac
     4081 tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc
     4141 ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc
     4201 ggcccgggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg
     4261 cgctggctgt cgacgctcga gatttccggc cggccttaag gcctcgaggg ccaattaatt
     4321 aactctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta
     4381 aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
     4441 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4501 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4561 taacgcgtcg agtgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4621 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4681 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4741 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4801 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4861 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4921 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4981 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5041 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5101 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5161 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5221 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     5281 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5341 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5401 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5461 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     5521 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5581 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5641 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5701 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5761 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5821 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5881 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5941 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6001 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6061 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6121 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6181 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6241 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6301 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6361 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6421 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6481 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6541 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6601 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6661 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6721 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6781 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
