LOCUS       sequence-328219-        6835 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 6835)
  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 6835)
  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..6835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(54..73)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        245..624
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        625..828
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     779..799
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     873..892
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        873..891
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1656..1885
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1868..1887
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(1957..2531,2532..2534,2535..2543)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2124..2141)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2351..2370
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3301..3414
                     /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             3430..4314
                     /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    4465..4586
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4502..4521)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4688..4704)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4688..4704)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4701..4723)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4712..4728
                     /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(4736..4742,4743..4760,4761..4766))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4781..4802
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4919..4936)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5090..5678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5170..5189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5849..6640,6641..6709))
                     /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     6472..6491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6710..6814)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
       61 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
      121 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      181 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      241 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      301 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      361 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      421 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      481 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      541 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      601 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      661 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      721 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      781 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      841 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      901 gcttggtacc gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc
      961 ggtatggatg tatgcgtccg tcttgccctg tggctcctct ggggactcct cctgcaccag
     1021 ggccagagcc tcagccatag tcacagtgag aaggcgacag gaaccagctc gggcgccggt
     1081 ttaaacgacc ctagcaaaga ctcaaaagct caggtgtccg ctgccgaggc tggcattact
     1141 ggaacatggt acaatcagct cgggagcacc tttattgtga ctgctggagc cgatggagcc
     1201 ctcaccggaa catacgaatc tgctgtggga aacgccgaat cacggtacgt cctcactggc
     1261 cgatacgata gtgcccctgc caccgacgga tctgggactg ccctgggatg gactgtcgct
     1321 tggaaaaaca actaccggaa tgctcattct gccacaacat ggagtggaca gtacgtggga
     1381 ggcgctgagg ctagaatcaa tacacagtgg ctgctcacat ctggcacaac cgaggcaaat
     1441 gcttggaaat ccaccctggt gggacatgac acattcacca aagtgaaacc ctccgccgct
     1501 tcaatcgatg ccgccaaaaa agccggagtc aacaacggca atcctctgga tgccgtccag
     1561 cagctcggac cgaaagatga actgtgagcg gccgcataga taactgatcc agtgtgctgg
     1621 aattaattcg ctgtctgcga gggccagctg ttggggtgag tactccctct caaaagcggg
     1681 catgacttct gcgctaagat tgtcagtttc caaaaacgag gaggatttga tattcacctg
     1741 gcccgcggtg atgcctttga gggtggccgc gtccatctgg tcagaaaaga caatcttttt
     1801 gttgtcaagc ttgaggtgtg gcaggcttga gatctggcca tacacttgag tgacaatgac
     1861 atccactttg cctttctctc cacaggtgtc cactcccagg tccaactgca ggtcgagcat
     1921 gcatctaggg cggccaattc cgcccctctc cccccccccc tctccctccc ccccccctaa
     1981 cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc
     2041 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac
     2101 gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt
     2161 gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg
     2221 caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata
     2281 agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga
     2341 aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt
     2401 accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc
     2461 gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac
     2521 acgatgataa gcttgccaca acccgggagg cgcgccatgt ctaccgagtc tatgattagg
     2581 gacgtggaac tggctgagga ggcactgccc aaaaaaaccg gcggaccaca gggctctagg
     2641 agatgtctgt ttctgtctct gttctctttt ctcatcgtgg ctggcgctac aacactcttc
     2701 tgtctgctcc atttcggcgt gattggacca cagcgagagg aatttccccg ggatctgtca
     2761 ctcatttcac cactggcaca ggctgtccga tcttcatctc ggactccatc cgacaaacct
     2821 gtcgcccatg tcgtcgccaa cccacaggcc gagggccagc tccagtggct caataggagg
     2881 gcaaacgctc tgctcgccaa tggcgtggaa ctccgggata accagctcgt cgtgcctagt
     2941 gagggactgt acctcatcta ctcccaggtg ctgtttaagg gccagggatg tccttctaca
     3001 catgtgctgc tcacacacac aatttcacgg atcgccgtgt cttaccagac taaagtcaat
     3061 ctgctctctg ccatcaaatc cccatgtcag cgggaaacac ctgagggcgc tgaggctaaa
     3121 ccttggtacg aacccatcta cctcggaggc gtgttccagc tggagaaggg cgatagactg
     3181 agtgctgaga tcaatcgacc cgactacctc gactttgccg aatctggcca ggtctacttt
     3241 ggcatcattg ctctgggcct cgagggccga gctcatggcg cacctaggcc tttgaattcc
     3301 atggacgaga agaccactgg ttggcgaggt ggacacgttg ttgaaggact ggctggggaa
     3361 cttgaacaac ttcgtgcacg actggagcat cacccacaag gtcaacgtga accacctgca
     3421 ggtggatccg aaatcggtac tggctttcca ttcgaccccc attatgtgga agtcctgggc
     3481 gagcgcatgc actacgtcga tgttggtccg cgcgatggca cccctgtgct gttcctgcac
     3541 ggtaacccga cctcctccta cgtgtggcgc aacatcatcc cgcatgttgc accgacccat
     3601 cgctgcattg ctccagacct gatcggtatg ggcaaatccg acaaaccaga cctgggttat
     3661 ttcttcgacg accacgtccg cttcatggat gccttcatcg aagccctggg tctggaagag
     3721 gtcgtcctgg tcattcacga ctggggctcc gctctgggtt tccactgggc caagcgcaat
     3781 ccagagcgcg tcaaaggtat tgcatttatg gagttcatcc gccctatccc gacctgggac
     3841 gaatggccag aatttgcccg cgagaccttc caggccttcc gcaccaccga cgtcggccgc
     3901 aagctgatca tcgatcagaa cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc
     3961 ccgctgactg aagtcgagat ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc
     4021 gagccactgt ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc
     4081 gcgctggtcg aagaatacat ggactggctg caccagtccc ctgtcccgaa gctgctgttc
     4141 tggggcaccc caggcgttct gatcccaccg gccgaagccg ctcgcctggc caaaagcctg
     4201 cctaactgca aggctgtgga catcggcccg ggtctgaatc tgctgcaaga agacaacccg
     4261 gacctgatcg gcagcgagat cgcgcgctgg ctgtctactc tggagatttc cggccggcct
     4321 taaggcctcg agggccaatt aattaactct agataactga tcataatcag ccataccaca
     4381 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     4441 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     4501 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4561 ttgtccaaac tcatcaatgt atcttaacgc gtcgagtgca ttctagttgt ggtttgtcca
     4621 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     4681 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4741 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4801 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4861 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4921 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4981 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5041 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5101 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5161 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5221 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5281 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5341 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5401 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5461 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5521 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5581 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5641 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5701 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5761 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5821 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5881 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5941 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6001 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6061 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6121 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6181 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6241 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6301 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6361 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6421 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6481 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6541 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6601 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6661 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6721 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6781 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgcc
//
