LOCUS       sequence-459265-        5847 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CleaveIL-6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5847)
  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 5847)
  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-0201 CMVTO-hIL6-rens.1
FEATURES             Location/Qualifiers
     source          1..5847
                     /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             3074..3103
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3159..3176)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3165..3389
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3561..3580)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3712..3759
                     /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             3767..4789
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3767..4789
                     /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    4919..5040
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4956..4975)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5010..5029
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5089..5105)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5089..5105)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5102..5124)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5113..5129
                     /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(5137..5143,5144..5161,5162..5167))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5182..5203
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5320..5337)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5491..5847,1..232))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5571..5590)
                     /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 agtcccccca ggcgaagatt ctaaagatgt ggctgctcca cacagacagc
     2521 cccttactag cagtgaacgt attgataaac agatcaggta catcctggac ggaatctcag
     2581 ccctaagaaa agagacatgc aataagtcca acatgtgtga gagttccaag gaggccctcg
     2641 ccgagaataa cctgaatcta cccaagatgg ccgagaagga cggttgtttc cagagtgggt
     2701 ttaacgaaga gacttgcctg gtgaaaatta taaccgggct gctggaattc gaagtgtacc
     2761 tggagtattt gcaaaatcgg ttcgagtcat ctgaagagca ggccagggct gtccagatgt
     2821 ctaccaaagt attaatccag ttcttgcaga agaaagctaa gaacctggat gcatatattc
     2881 acccattcca tctggttatt cataacgagt cgataacaac tcctgacccc accacaaacg
     2941 cgagcctgct tacaaagctc caggcgcaga atcagtggtt acaagacatg acgacccatc
     3001 tgattctccg atcatttaag gaatttttgc aatcctccct ccgggcactg cgccaaatga
     3061 ccggttaggg cccgaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca
     3121 tcatcataat catcattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     3181 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3241 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3301 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3361 gcatgctggg gatgcggtgg gctctatgga gatctatcga tgttctgggg atgcggtggg
     3421 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3481 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3541 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3601 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3661 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct
     3721 attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact
     3781 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     3841 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     3901 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     3961 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     4021 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     4081 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     4141 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     4201 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     4261 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     4321 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     4381 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     4441 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     4501 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     4561 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     4621 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     4681 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     4741 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg
     4801 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4861 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     4921 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4981 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5041 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5101 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5161 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5221 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5281 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5341 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5401 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5461 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5521 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5581 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5641 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5701 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5761 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5821 cgacttatcg ccactggcag cagccac
//
