LOCUS       sequence-459219-        5724 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutant human IL-10 monomer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5724)
  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 5724)
  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-0045 CMVTO-mono-hIL10 (R5A11M)
FEATURES             Location/Qualifiers
     source          1..5724
                     /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             2951..2980
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3036..3053)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3042..3266
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3438..3457)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3589..3636
                     /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             3644..4666
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3644..4666
                     /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    4796..4917
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4833..4852)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4887..4906
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4966..4982)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4966..4982)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4979..5001)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4990..5006
                     /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(5014..5020,5021..5038,5039..5044))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5059..5080
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5197..5214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5368..5724,1..232))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5448..5467)
                     /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 caccatgcac tcctccgcgc
     2401 tcttgtgttg cctagtttta ttgaccggag tccgcgcatc acctgggcaa ggcactcaga
     2461 gtgaaaattc ttgcactcac ttccctggaa ttctccccaa catgctcaga gacctgcgag
     2521 atgctttctc tagagtcaaa acgttctttc aaatgaagga ccagctagat aatcttttgc
     2581 tgaaagagtc ccttctcgag gattttaaag gctatctggg ctgtcaggct cttagcgaga
     2641 tgatacagtt ctacctcgag gaggtgatgc cacaggctga gaaccaagac ccagacatta
     2701 aagcccatgt aatctcactg ggggaaaacc tgaacacatt acgcctgcgg ctgcggaggt
     2761 gccataggct gctgccttgt gaaaatggtg ggggcagtgg aggcaagagc aaggccgtgg
     2821 aacaggtgaa gaatgccttt aataagctgc aggaaaaggg gatctacaag gcaatgagcg
     2881 agtttgatat attcatcaac tatatcgagg cctacatgac catgaaaatt cgtaacaccg
     2941 gttagggccc gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca
     3001 tcataatcat cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     3061 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3121 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3181 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3241 tgctggggat gcggtgggct ctatggagat ctatcgatgt tctggggatg cggtgggctc
     3301 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3361 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3421 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3481 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3541 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt
     3601 ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac
     3661 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     3721 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     3781 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     3841 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct
     3901 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     3961 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     4021 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     4081 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     4141 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     4201 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     4261 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     4321 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     4381 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     4441 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     4501 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     4561 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     4621 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga
     4681 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4741 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4801 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4861 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4921 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4981 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5041 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5101 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5161 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5221 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5281 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5341 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5401 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5461 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5521 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5581 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5641 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5701 cttatcgcca ctggcagcag ccac
//
