LOCUS       sequence-459334-        6456 bp    DNA     circular SYN 15-APR-2025
DEFINITION  CageIL-10-monomer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6456)
  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 6456)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-0091 CMVTO-mono-hIL10-R5A11M-rens-3GS-IL10Ra
FEATURES             Location/Qualifiers
     source          1..6456
                     /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             3683..3712
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3768..3785)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3774..3998
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4170..4189)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4321..4368
                     /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             4376..5398
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4376..5398
                     /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    5528..5649
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5565..5584)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5619..5638
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5698..5714)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5698..5714)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5711..5733)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5722..5738
                     /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(5746..5752,5753..5770,5771..5776))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5791..5812
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5929..5946)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6100..6456,1..232))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6180..6199)
                     /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 gctatattca cccattccat ctggttattc ataacgagtc gggcggtggg ggatctggtg
     3001 gagggggatc aggaggtggc ggatcccatg gcactgagct tccatctcct ccttctgtat
     3061 ggttcgaagc agagttcttc catcacattc tgcattggac tcccattcca aaccaatcgg
     3121 agagcacctg ctacgaagtg gctctgttaa gatacggcat tgagtcttgg aacagcatca
     3181 gcaactgctc acaaacgctc tcttatgatt tgaccgcggt gacacttgac ctctaccact
     3241 caaacggcta tagagcccgg gttcgagccg tggatggctc ccgacactcc aactggaccg
     3301 tcactaatac acggtttagc gtggacgagg tgaccctgac agtgggaagt gtgaacctcg
     3361 aaattcataa cgggtttatt ctgggaaaaa tacagctgcc tcgtccaaag atggcaccag
     3421 ccaacgacac gtacgaaagc attttcagcc acttccggga gtacgaaatc gcaatccgca
     3481 aggtaccggg taattttact tttacccaca agaaggttaa gcacgaaaac ttcagcttgt
     3541 taacctctgg cgaggtcgga gagttttgcg tgcaagtgaa gcctagcgtc gcttcacgct
     3601 ccaataaagg tatgtggagt aaagaggagt gcatcagtct cacaaggcag tattttacgg
     3661 tcaccaatac cggttagggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc
     3721 cgtcgaccat catcataatc atcattgagt ttaaacccgc tgatcagcct cgactgtgcc
     3781 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3841 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3901 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3961 caatagcagg catgctgggg atgcggtggg ctctatggag atctatcgat gttctgggga
     4021 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     4081 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4141 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4201 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4261 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtaccta
     4321 gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct tggccaaaaa
     4381 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc
     4441 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg
     4501 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt
     4561 ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt
     4621 cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct
     4681 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc
     4741 tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca
     4801 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca
     4861 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct
     4921 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa
     4981 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg
     5041 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga
     5101 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg
     5161 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt
     5221 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac
     5281 tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga
     5341 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagca
     5401 cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     5461 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     5521 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     5581 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     5641 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     5701 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5761 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5821 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5881 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5941 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     6001 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     6061 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6121 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6181 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6241 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6301 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6361 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6421 ggtaagacac gacttatcgc cactggcagc agccac
//
