LOCUS       sequence-363891-        5240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the brine shrimp protein AfrLEA3m. 
            This is one of the top performing targets in our apoptosis assay (it
            protected from apoptosis)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5240)
  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 5240)
  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_AfrLEA3m (pBS0911)
FEATURES             Location/Qualifiers
     source          1..5240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(54..73)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        245..624
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        625..828
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     779..799
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     873..892
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        873..891
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      909..918
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1218..1241
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1258..1275)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1264..1488
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1534..1962
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1621..1640)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1831..1852
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1971..1991)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1976..2305
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2156..2291
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2218..2237
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2353..2400
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2419..2793
                     /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    2923..3044
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2960..2979)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3014..3033
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3093..3109)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3093..3109)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3106..3128)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3117..3133
                     /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(3141..3147,3148..3165,3166..3171))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3324..3341)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3495..4083)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3575..3594)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4254..5045,5046..5114))
                     /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     4877..4896
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5115..5219)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
       61 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
      121 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      181 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      241 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      301 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      361 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      421 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      481 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      541 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      601 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      661 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      721 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      781 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      841 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      901 gctggctagc caccatggag ctgagcagca gcaagctgaa caggaacatc ttcaagagga
      961 gcagcagcgt gctgcactgg cacttctaca ggctgttcgg caagaagtac cccagcaaga
     1021 tgagcgagca gggcaagctg agcaggcagg aggccggcca aaggggagga caagccagag
     1081 cagaacaatt gggccatgaa ggctacgtgg agatgggcag gaagggcggc caagccagag
     1141 ccgaacaatt gggtcatgaa ggataccagg agatgggcca gaagggcggc ctggccaggc
     1201 agcagtgagg atctggggac tacaaggacg acgacgacaa gtgaacccgc tgatcagcct
     1261 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     1321 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     1381 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     1441 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     1501 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     1561 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1621 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1681 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1741 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1801 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1861 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1921 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     1981 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2041 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2101 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     2161 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2221 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     2281 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     2341 tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     2401 aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc
     2461 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg
     2521 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
     2581 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
     2641 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
     2701 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
     2761 tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc
     2821 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     2881 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     2941 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     3001 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     3061 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3121 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3181 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3241 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3301 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3361 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3421 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3481 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3541 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3601 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3661 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3721 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3781 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3841 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3901 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     3961 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4021 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4081 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4141 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4201 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4261 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4321 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4381 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4441 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4501 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4561 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4621 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4681 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4741 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4801 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4861 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4921 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4981 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5041 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5101 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5161 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     5221 acatttcccc gaaaagtgcc
//
