LOCUS       sequence-363969-        6760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the brine shrimp protein AfrLEA3m 
            attached to 2xFKBP. The FKBP can be used for inducible dimerization 
            and condensate formation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6760)
  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 6760)
  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: AfrLEA3m_mCh-2xFKBP (pBS1137)
FEATURES             Location/Qualifiers
     source          1..6760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(42..61)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        233..612
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        613..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     767..787
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    818..836
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    839..857
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     867..891
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1000..1009
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2038..2358
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             2365..2685
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             2692..2718
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(2766..2783)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2772..2996
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3042..3470
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3129..3148)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3339..3360
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3479..3499)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3484..3813
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3664..3799
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3726..3745
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3861..3908
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3927..4301
                     /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    4431..4552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4468..4487)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4522..4541
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4601..4617)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4601..4617)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4614..4636)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4625..4641
                     /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(4649..4655,4656..4673,4674..4679))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4694..4715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4832..4849)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5003..5591)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5083..5102)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5762..6553,6554..6622))
                     /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     6385..6404
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6623..6727)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
       61 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
      121 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
      181 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
      241 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      301 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      361 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      421 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      481 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      541 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      601 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      661 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      721 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      781 aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga tagagatctc
      841 cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat cgcctggaga
      901 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccggact
      961 ctagcgttta aacttaagct tggtaccgag ctcggatccg ccaccatgga gctgagcagc
     1021 agcaagctga acaggaacat cttcaagagg agcagcagcg tgctgcactg gcacttctac
     1081 aggctgttcg gcaagaagta ccccagcaag atgagcgagc agggcaagct gagcaggcag
     1141 gaggccggcc aaaggggagg acaagccaga gcagaacaat tgggccatga aggctacgtg
     1201 gagatgggca ggaagggcgg ccaagccaga gccgaacaat tgggtcatga aggataccag
     1261 gagatgggcc agaagggcgg cctggccagg cagcagtccg gaggcggcgg atccggcgga
     1321 atggtgtcta aaggcgagga ggataacatg gctataatca aggaatttat gaggttcaag
     1381 gtgcatatgg agggatctgt gaacggtcac gagttcgaaa tcgagggcga aggcgagggg
     1441 cgaccctatg aagggacaca gacggccaaa ctgaaggtga ccaagggtgg ccccctgcct
     1501 ttcgcctggg acatcctgtc accccagttt atgtacggct ctaaggctta cgttaagcac
     1561 cctgccgata ttccggacta cttgaaactg agcttccctg aagggttcaa gtgggaacgg
     1621 gtaatgaatt ttgaggatgg cggtgtagtc acagttaccc aggacagctc tcttcaggac
     1681 ggagaattta tctataaggt taaacttcgg ggcactaact tcccatcaga cggccccgtg
     1741 atgcagaaga aaactatggg ctgggaagcc agctcagaac gcatgtatcc cgaggacgga
     1801 gcactgaagg gagaaatcaa gcagcgactc aagctgaagg atgggggaca ttatgatgct
     1861 gaggtcaaaa ccacctataa ggccaagaaa cccgttcagc ttcctggggc atacaacgtg
     1921 aatatcaaac tggatattac atcccataac gaagactata ccatcgtgga acagtacgag
     1981 cgggccgagg gcagacatag cacagggggc atggatgaat tgtacaaggc gactagaggc
     2041 gtccaagtcg aaaccattag tcccggcgat ggcagaacat ttcctaaaag gggacaaaca
     2101 tgtgtcgtcc attatacagg catgttggag gacggcaaaa aggtggacag tagtagagat
     2161 cgcaataaac ctttcaaatt catgttggga aaacaagaag tcattagggg atgggaggag
     2221 ggcgtggctc aaatgtccgt cggccaacgc gctaagctca ccatcagccc cgactacgca
     2281 tacggcgcta ccggacatcc cggaattatt ccccctcacg ctaccttggt gtttgacgtc
     2341 gaactgttga agctcgagac tagaggagtg caggtggaga ctatctcccc aggagacggg
     2401 cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat
     2461 ggaaagaaag ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag
     2521 caggaggtga tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc
     2581 aaactgacta tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca
     2641 ccacatgcca ctctcgtctt cgatgtggag cttctaaaac tggaaactag ttatccgtac
     2701 gacgtaccag actacgcata agcggccgct cgagtctaga gggcccgttt aaacccgctg
     2761 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2821 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2881 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2941 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     3001 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     3061 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3121 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3181 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3241 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3301 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3361 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3421 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     3481 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3541 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3601 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3661 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3721 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3781 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3841 ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat
     3901 aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca
     3961 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
     4021 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
     4081 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
     4141 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
     4201 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
     4261 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg
     4321 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4381 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4441 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4501 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4561 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4621 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4681 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4741 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4801 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4861 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4921 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4981 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5041 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5101 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5161 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5221 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5281 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5341 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5401 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5461 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5521 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5581 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5641 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5701 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5761 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5821 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5881 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5941 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6001 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6061 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6121 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6181 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6241 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6301 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6361 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6421 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6481 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6541 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6601 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6661 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6721 ttccgcgcac atttccccga aaagtgccac ctgacgtcga
//
