LOCUS       sequence-363890-        6041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the brine shrimp protein AfrLEA2. 
            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 6041)
  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 6041)
  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: pST_AfrLEA2 (pBS0910)
FEATURES             Location/Qualifiers
     source          1..6041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..58)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        230..609
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        610..813
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     764..784
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     858..877
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        858..876
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2004..2027
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2044..2061)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2050..2274
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2320..2748
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2407..2426)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2617..2638
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2757..2777)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2762..3091
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2942..3077
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3004..3023
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3139..3186
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3205..3579
                     /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    3709..3830
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3746..3765)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3800..3819
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3879..3895)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3879..3895)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3892..3914)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3903..3919
                     /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(3927..3933,3934..3951,3952..3957))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4110..4127)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4281..4869)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4361..4380)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5040..5831,5832..5900))
                     /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     5663..5682
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5901..6005)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
       61 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
      121 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
      181 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      241 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      301 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      361 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      421 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      481 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      541 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      601 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      661 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      721 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      781 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      841 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagccacca
      901 tgcctaaggc tgctgccaag ggcatcggag aaaccgtcaa ggccgacgcc gacgttgtgg
      961 agggtatggc atcaaccgga tatgaaaaat tgaagagcgc cttcggcatc gcttccaata
     1021 agaccaagga cgctgcagag aacgtggctg agtctgccag ggcaaccaaa gattatactg
     1081 tggatagtgc caagtccgca tacgacaaaa ccgtggatag caccaagtcc gcctacgaca
     1141 agaccaccga ctccgccaag agcgtgcatg actccaccgc cgacactgca aagtcagctt
     1201 ataataaggc caccgaaaca ctggggagcg cttacgataa gaccaaggac accgcccaga
     1261 gcacttacga tcagcaagtg ggtgccgcac actccgccta cgacaagacc gctgaggcca
     1321 ccaagagcgc ctacgacaag accgccgacg ccgcccactc cgtctacaac aagaccggag
     1381 acgccggcaa acaggcttac gacagcacca aggaggccgc caggagtacc ggcaaaagca
     1441 tctctgatgc cgcttatttc acaggtaagg gggccgagcg gcagggcgat caggtgaagt
     1501 ctgaactgcc ttcttactcc ccaagcagca gcggagagaa gctggctcag cacctggtga
     1561 aaagcgagaa agaggggaag aagctcaccg aggaggccct caaggatagg gacctgtcac
     1621 aggtgccagg tttcagatcc gtgaaaaagg ctcacgaacc cgacgccaag gaggatatct
     1681 ctgccgtgga cttcgcctcc gccagtccct cccagagaaa ggtggctgac accgaggggg
     1741 tgtggagctc accagtggac agacaggaga gcagattctt cagcgatctc gccggcaaga
     1801 tcggagatat gctggggggc ggaaagatca acgcaattca gacccctgag gaaatggatc
     1861 acgagagatt gattcataag agtagccaga gccaggtggc aggcaacgtt cctgggagag
     1921 ctaagacagc ctggacccct gaggacagaa tcatcctgca ccaggagcgg ttccccaaag
     1981 agaaccccga gtgaggatct ggggactaca aggacgacga cgacaagtga acccgctgat
     2041 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2101 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2161 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2221 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     2281 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     2341 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2401 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2461 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2521 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2581 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2641 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2701 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     2761 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2821 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     2881 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     2941 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3001 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     3061 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     3121 cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     3181 tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc ggtgctcacc
     3241 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac
     3301 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg
     3361 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac
     3421 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg
     3481 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc
     3541 ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat
     3601 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3661 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     3721 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3781 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     3841 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     3901 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     3961 tggggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4021 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4081 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4141 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4201 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4261 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4321 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4381 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4441 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4501 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4561 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4621 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4681 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4741 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4801 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4861 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4921 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4981 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5041 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5101 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5161 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5221 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5281 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5341 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5401 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5461 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5521 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5581 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5641 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5701 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5761 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5821 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5881 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5941 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6001 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg g
//
