LOCUS       sequence-459262-        5643 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CleaveIL-2_loop2_del.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5643)
  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 5643)
  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-0212 CMVTO-hIL2-rens-del.3
FEATURES             Location/Qualifiers
     source          1..5643
                     /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             3037..3066
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3122..3139)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3128..3352
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3524..3543)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3675..3722
                     /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             3730..4752
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3730..4752
                     /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    4882..5003
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4919..4938)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4973..4992
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5052..5068)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5052..5068)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5065..5087)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5076..5092
                     /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(5100..5106,5107..5124,5125..5130))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5145..5166
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5283..5300)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5454..5643,1..399))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5534..5553)
                     /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 tagcgcacct acttcaagtt ctacaaagaa
     2641 aacacagcta caactggagc atttactgct ggatttacag atgattttga atggaattaa
     2701 taattacaag aatcccaaac tcaccaggat gctcacattt aagttttaca tgcccaagaa
     2761 ggccacagaa ctgaaacatc ttcagtgtct agaagaagaa ctcaaacctc tggaggaagt
     2821 gctaaattta gcttatattc acccattcca tctggttatt cataacgagt cgcccaggga
     2881 cttaatcagc aatatcaacg taatagttct ggaactaaag ggatctgaaa caacattcat
     2941 gtgtgaatat gctgatgaga cagcaaccat tgtagaattt ctgaacagat ggattacctt
     3001 ttgtcaaagc atcatctcaa cactgactta gggcccgaac aaaaactcat ctcagaagag
     3061 gatctgaata gcgccgtcga ccatcatcat aatcatcatt gagtttaaac ccgctgatca
     3121 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     3181 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     3241 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     3301 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggagatctat
     3361 cgatgttctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     3421 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3481 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3541 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3601 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3661 ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact
     3721 tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
     3781 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
     3841 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
     3901 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
     3961 gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
     4021 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
     4081 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     4141 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     4201 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     4261 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     4321 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     4381 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     4441 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     4501 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     4561 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     4621 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     4681 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
     4741 gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4801 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4861 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4921 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4981 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5041 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5101 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5161 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5221 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5281 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5341 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5401 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5461 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5521 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5581 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5641 ctt
//
