LOCUS       sequence-365634-        4554 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the tardigrade protein CAHS2. This 
            is one of the bottom performing targets in our apoptosis assay (it 
            induced apoptosis)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4554)
  AUTHORS   Veling MT, Nguyen DT, Thadani NN, Oster ME, Rollins NJ, Brock KP, 
            Bethel NP, Lim S, Baker D, Way JC, Marks DS, Chang RL, Silver PA
  TITLE     Natural and Designed Proteins Inspired by Extremotolerant Organisms 
            Can Form Condensates and Attenuate Apoptosis in Human Cells.
  JOURNAL   ACS Synth Biol. 2022 Mar 18;11(3):1292-1302. doi: 
            10.1021/acssynbio.1c00572. Epub 2022 Feb 18.
  PUBMED    35176859
REFERENCE   2  (bases 1 to 4554)
  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"; doi: 
            "10.1021/acssynbio.1c00572"; journalName: "ACS Synth Biol"; date: 
            "2022-03-18- 18"; volume: "11"; issue: "3"; pages: "1292-1302"
COMMENT     Sequence Label: CAHS2 (pBS0249)
FEATURES             Location/Qualifiers
     source          1..4554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(51..70)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        242..621
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        622..825
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     776..796
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     870..889
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        870..888
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      905..914
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             914..937
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        1664..1711
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             1730..2104
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    2234..2355
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2271..2290)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2325..2344
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2404..2420)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2404..2420)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2417..2439)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2428..2444
                     /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(2452..2458,2459..2476,2477..2482))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(2635..2652)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2806..3394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2886..2905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3565..4356,4357..4425))
                     /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     4188..4207
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4426..4530)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
       61 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
      121 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
      181 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
      241 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      301 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      361 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      421 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      481 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      541 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      601 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      661 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      721 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      781 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
      841 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccgaatt
      901 cgccgccacc atggattaca aggatgatga tgataagatg gaggccatga atatgaatat
      961 cccccgcgat gccatgtttg ttccgccacc ggaatctgag caaaatgggt atcatgagaa
     1021 gtcagaagtt cagcaaacaa gttatatgca gagtcaagtc aaagtgccac attataattt
     1081 cccgacacca tattttacga cttccttttc tgcgcaagag ctgctgggcg aagggtttca
     1141 agcctcaatt tcccgtattt cagccgttac ggaagacatg cagagcatgg aaatcccgga
     1201 gttcgttgaa gaggcccgcc gtgattacgc agccaaaaca cgtgaaaatg agatgctggg
     1261 gcaacaatat gaaaaagagc tggaacgtaa gtccgaagcc taccgcaaac atcaggaagt
     1321 agaggccgac aaaatccgca aagaacttga aaaacagcat atgcgtgata ttgaatttcg
     1381 gaaagaaatt gcagaactgg cgattgagaa ccaaaaacgt atgatcgatc ttgaatgccg
     1441 ctatgcaaaa aaagacatgg accgggaacg cacaaaagtt cgtatgatgc tggagcaaca
     1501 gaaattccat agtgatatcc aggtaaatct ggattcttct gcggctggga ccgagagcgg
     1561 aggtcatgta gtgagccagt ctgaaaagtt caccgaacgt aaccgcgaga tgaaacgcta
     1621 agggtgtttg tggttttgtt gtgtggtatt gtgatcagca cgtgttgaca attaatcatc
     1681 ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt
     1741 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac
     1801 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga
     1861 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc
     1921 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac
     1981 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg
     2041 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga
     2101 ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     2161 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     2221 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2281 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     2341 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     2401 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2461 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac ttacattaat
     2521 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2581 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2641 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2701 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2761 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2821 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2881 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2941 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3001 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3061 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3121 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3181 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3241 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3301 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3361 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3421 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3481 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3541 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3601 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3661 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3721 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3781 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3841 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3901 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3961 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4021 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4081 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4141 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4201 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4261 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4321 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4381 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4441 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4501 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacc
//
