LOCUS       sequence-459373-        5682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CleaveIL-2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5682)
  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 5682)
  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-0234 CMVTO-sumIL2-rensGS.3
FEATURES             Location/Qualifiers
     source          1..5682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(570..1361,1362..1430))
                     /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     1193..1212
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1431..1535)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1610..1629)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1796..2175
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2176..2379
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2330..2350
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2381..2399
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2402..2420
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2430..2454
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             3076..3105
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3161..3178)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3167..3391
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3563..3582)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3714..3761
                     /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             3769..4791
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3769..4791
                     /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    4921..5042
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4958..4977)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5012..5031
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5091..5107)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5091..5107)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5104..5126)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5115..5131
                     /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(5139..5145,5146..5163,5164..5169))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5184..5205
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5322..5339)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5493..5682,1..399))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5573..5592)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
       61 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
      121 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
      181 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
      241 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
      301 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
      361 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
      421 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
      481 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
      541 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
      601 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
      661 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
      721 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
      781 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
      841 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
      901 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
      961 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     1021 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     1081 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     1141 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     1201 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     1261 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     1321 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     1381 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     1441 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     1501 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     1561 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     1621 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     1681 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     1741 atctgcttag ggttaggcgt tttgcgctgc ttcgtacggg ccagatatac gcgttgacat
     1801 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     1861 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     1921 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     1981 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     2041 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     2101 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     2161 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
     2221 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
     2281 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
     2341 ggtaggcgtg tacggtggga ggtctatata agcagagctc tccctatcag tgatagagat
     2401 ctccctatca gtgatagaga tcgtcgacga gctcgtttag tgaaccgtca gatcgcctgg
     2461 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
     2521 actctagcgt ttaaacttaa gcttcgccac catgtacagg atgcaactcc tgtcttgcat
     2581 tgcactaagt cttgcacttg tcacaaacgc tagcgctcca acctccagct ctacaaaaaa
     2641 gactcagctg cagctggagc acctgctgct cgacctacaa atgatactca acggaatcaa
     2701 caactataag aatcctaagc tgacgcggat gctgaccgcc aagttttaca tgcccaaaaa
     2761 ggcgaccgag ctgaagcatt tgcagtgtct ggaggaagag ctcaagcctt tggaggaggt
     2821 gcttaatctc gcccaaagca aaaattatat tcacccattc catctggtta ttcataacga
     2881 gtcgggcggt gggggatcct tccactttga tccgcgggac gtcgtgtcaa atatcaacgt
     2941 ctttgtgctg gaactgaaag gctctgagac aacctttatg tgtgaatatg ccgatgaaac
     3001 tgctaccatc gttgagtttt taaatcgatg gataacattt tgccagagta taatttctac
     3061 tttgacctag ggcccgaaca aaaactcatc tcagaagagg atctgaatag cgccgtcgac
     3121 catcatcata atcatcattg agtttaaacc cgctgatcag cctcgactgt gccttctagt
     3181 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3241 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3301 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     3361 aggcatgctg gggatgcggt gggctctatg gagatctatc gatgttctgg ggatgcggtg
     3421 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     3481 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3541 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3601 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3661 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc
     3721 ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa
     3781 ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg
     3841 atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga
     3901 tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg
     3961 cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag
     4021 agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa
     4081 accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc
     4141 gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact
     4201 acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg
     4261 atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc
     4321 gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg
     4381 acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc
     4441 caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag
     4501 acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat
     4561 atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat
     4621 gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg
     4681 cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc
     4741 gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta
     4801 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     4861 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     4921 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4981 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5041 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     5101 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5161 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5221 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5281 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5341 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5401 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5461 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5521 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5581 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5641 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tt
//
