LOCUS       sequence-459404-        8784 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multi-cageIL-6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8784)
  AUTHORS   Aldrete CA, Call CC, Sant'Anna LE, Vlahos AE, Pei J, Cong Q, Gao XJ
  TITLE     Orthogonalized human protease control of secreted signals.
  JOURNAL   Nat Chem Biol. 2025 Jan 15. doi: 10.1038/s41589-024-01831-x.
  PUBMED    39814991
REFERENCE   2  (bases 1 to 8784)
  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"; journalName: "Nat Chem 
            Biol. 2025 Jan 15. doi: 10.1038/s41589-024-01831-x."
COMMENT     Sequence Label: CA-0216 CMVTO-hIL6Ra_M4-rens-hIL6-rens-IL6Rb
FEATURES             Location/Qualifiers
     source          1..8784
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(403..1194,1195..1263))
                     /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     1026..1045
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1264..1368)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1443..1462)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1629..2008
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2009..2212
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2163..2183
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2214..2232
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2235..2253
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2263..2287
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             6011..6040
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(6096..6113)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6102..6326
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6498..6517)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    6649..6696
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             6704..7726
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             6704..7726
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    7856..7977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7893..7912)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7947..7966
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8026..8042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8026..8042)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8039..8061)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8050..8066
                     /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(8074..8080,8081..8098,8099..8104))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8119..8140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8257..8274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(8428..8784,1..232))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8508..8527)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
       61 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      121 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      181 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      241 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      301 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      361 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      421 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
      481 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
      541 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
      601 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
      661 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
      721 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
      781 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
      841 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
      901 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
      961 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1021 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1081 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1141 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1201 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1261 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1321 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1381 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     1441 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     1501 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     1561 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgtac gggccagata
     1621 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     1681 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     1741 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     1801 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     1861 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     1921 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     1981 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     2041 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     2101 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     2161 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat
     2221 cagtgataga gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg
     2281 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg
     2341 atccagcctc cggactctag cgtttaaact taagcttcgc caccatgaac agcttctcta
     2401 ccagcgcttt tggacccgtg gccttttcgt tgggcctgct tctcgttctt cctgccgcat
     2461 tccctgcacc acttgcgccg agaagatgcc ccgcccaaga ggtggcacgg ggagttctga
     2521 ccagtctccc aggcgattct gtgactctga cctgccctgg tgtagaaccg gaagacaatg
     2581 caaccgtcca ctgggtgttg agaaagccag cagcgggaag ccatccctcc aggtgggccg
     2641 gcatgggacg acgcctgctg ctgaggagtg tgcagctaca cgattctggt aactactctt
     2701 gttatagggc cggccggccg gctggcaccg tccatttact cgtggatgtg cctcccgagg
     2761 agccccaatt gagttgcttt cgaaaaagcc cactgtccaa tgtagtctgc gagtggggtc
     2821 cacggtcgac cccctccctc acaacgaaag cggtcttact tgttaggaag ttccagaaca
     2881 gccctgctga ggacttccag gaaccttgtc agtacagtca agagtctcag aagtttagct
     2941 gtcaattggc agtcccagag ggggacagca gcttttatat cgtatcaatg tgcgtggcct
     3001 cctccgtggg gagtaagttc tccaagaccc agactttcca gggctgtggc attcttcagc
     3061 ctgacccacc agccaacatt actgtgaccg ccgtagacag agatccccgt tggctgagcg
     3121 ttacttggca agacccccat agttggaact catctttcta tcggctccgc tttaagctcc
     3181 gttaccgcgc cgaacgcagc aagacattta cgacatggat ggtcaaagat cttcagcacc
     3241 actgtgtgat atctgtggct tggtccgggc tgaggcacgt tgtccagctg agagcccagg
     3301 aggaatttgg acagggggag tggtccgagt ggtcaccaga agctatgggg actccttgga
     3361 cagaatctcg atcccccccc gctgagaatg aagtgtcaac ccccatgcag gcactgacaa
     3421 caaataaaga tgacgataat atcctattcc gggactcagc caacgctact tcgttgcctg
     3481 ttcaggacag ctcttcagtg cctctgccta ccggtggcgg cggtagcgga ggcgggggta
     3541 gctatattca cccattccat ctggttattc ataacgagtc gggtaccgtc cccccaggcg
     3601 aagattctaa agatgtggct gctccacaca gacagcccct tactagcagt gaacgtattg
     3661 ataaacagat caggtacatc ctggacggaa tctcagccct aagaaaagag acatgcaata
     3721 agtccaacat gtgtgagagt tccaaggagg ccctcgccga gaataacctg aatctaccca
     3781 agatggccga gaaggacggt tgtttccaga gtgggtttaa cgaagagact tgcctggtga
     3841 aaattataac cgggctgctg gaattcgaag tgtacctgga gtatttgcaa aatcggttcg
     3901 agtcatctga agagcaggcc agggctgtcc agatgtctac caaagtatta atccagttct
     3961 tgcagaagaa agctaagaac ctggatgcaa taacaactcc tgaccccacc acaaacgcga
     4021 gcctgcttac aaagctccag gcgcagaatc agtggttaca agacatgacg acccatctga
     4081 ttctccgatc atttaaggaa tttttgcaat cctccctccg ggcactgcgc caaatgaccg
     4141 gtggcggcgg tagcggatat attcacccat tccatctggt tattcataac gagtcgggcg
     4201 gtggtaccgg cgagctgctg gatccttgcg ggtacatctc gcccgaaagc ccagttgtcc
     4261 agctacactc aaattttacc gcggtgtgcg tgctgaagga aaaatgcatg gactattttc
     4321 acgtgaacgc gaactatatt gtctggaaga cgaaccactt tactataccc aaggaacagt
     4381 acacaatcat taaccgcacc gcaagttcgg tgactttcac tgacattgca tctctgaata
     4441 tccagctgac ctgcaacatt ctgacttttg gccagttaga acaaaacgtg tatgggatca
     4501 ctatcataag cggccttcca cctgagaagc caaagaatct gtcatgtatc gtcaacgagg
     4561 gcaagaagat gcgctgcgaa tgggacggcg ggcgtgagac acacctagag acgaatttca
     4621 ccctcaaaag tgagtgggct actcataaat ttgcagattg caaggcaaag agggacacac
     4681 ctacctcttg cactgtcgac tactccaccg tatactttgt taacatcgag gtgtgggttg
     4741 aagctgaaaa cgccctgggc aaggtgacct ctgatcacat caactttgat ccagtttaca
     4801 aagtgaaacc taaccctccg cacaatctgt cagtgatcaa ctccgaagaa ctcagtagca
     4861 ttctgaaact cacgtggact aatccctcaa ttaaaagtgt cataatttta aaatacaata
     4921 tccagtaccg gaccaaggat gccagtacgt ggagccaaat tcctcctgag gacaccgcta
     4981 gtactcgcag ctcatttact gtgcaggacc tgaagccttt cacagagtat gtcttcagaa
     5041 tcagatgtat gaaggaggac ggaaagggat actggtctga ctggtccgag gaggcctctg
     5101 gaatcaccta tgaagatcgg cccagcaaag ccccatcctt ctggtacaaa atcgatcctt
     5161 cccataccca aggatatcgg accgtgcagt tggtgtggaa gacattgccc cccttcgaag
     5221 ccaatgggaa gatccttgac tacgaagtga cattgacaag gtggaaaagt cacctgcaga
     5281 attataccgt caatgctact aagctgacag taaatctcac taatgatagg tacttggcaa
     5341 cgcttacagt gcgaaatttg gttggcaagt ccgacgccgc tgtcctgaca atccccgctt
     5401 gcgacttcca ggctacccac ccagtgatgg acttgaaggc attccctaag gataacatgc
     5461 tgtgggtaga gtggaccacg cctcgggaga gcgtgaagaa atatatcctc gagtggtgtg
     5521 tgctgtccga caaagccccg tgcattaccg actggcagca agaggatggg actgttcaca
     5581 gaacatacct cagaggcaac ttggccgaaa gcaaatgtta tctaataaca gtgaccccgg
     5641 tttacgccga cggtccaggc agtccagagt cgattaaggc ctatctgaaa caggctcctc
     5701 cttctaaggg acctacagtt aggacaaaga aagtgggaaa gaatgaggcg gttctcgagt
     5761 gggaccagct gccggtggat gtccagaatg gctttatccg aaactatacg atcttttaca
     5821 gaacaatcat tgggaacgaa acagccgtta acgtagactc cagtcatacc gagtacacac
     5881 taagctcact tacttctgac accctctata tggtccgaat ggccgcctac actgacgagg
     5941 gaggaaaaga cggaccagaa ttcaccttta ctacgccaaa atttgcgcag ggcgagatcg
     6001 aatagggccc gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca
     6061 tcataatcat cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     6121 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     6181 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     6241 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     6301 tgctggggat gcggtgggct ctatggagat ctatcgatgt tctggggatg cggtgggctc
     6361 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     6421 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     6481 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     6541 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     6601 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt
     6661 ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac
     6721 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     6781 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     6841 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     6901 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct
     6961 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     7021 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     7081 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     7141 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     7201 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     7261 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     7321 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     7381 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     7441 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     7501 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     7561 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     7621 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     7681 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga
     7741 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     7801 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     7861 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     7921 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     7981 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     8041 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     8101 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     8161 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     8221 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     8281 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     8341 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     8401 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     8461 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     8521 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     8581 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     8641 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     8701 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     8761 cttatcgcca ctggcagcag ccac
//
