LOCUS       sequence-440207-        6424 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Biosensor chain detecting rapamycin and responding with split 
            Nanoluciferase reconstitution. FRB ectodomain, human Glycophorin A 
            (GPA) scaffold, 20 GS linker, split Nanoluciferase fragment 114..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6424)
  AUTHORS   Dolberg TB, Gunnels TF, Ling T, Sarnese KA, Crispino JD, Leonard JN
  TITLE     Building Synthetic Biosensors Using Red Blood Cell Proteins.
  JOURNAL   ACS Synth Biol. 2024 Apr 19;13(4):1273-1289. doi: 
            10.1021/acssynbio.3c00754. Epub 2024 Mar 27.
  PUBMED    38536408
REFERENCE   2  (bases 1 to 6424)
  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.3c00754"; journalName: "ACS Synth Biol"; date: 
            "2024-04-19- 19"; volume: "13"; issue: "4"; pages: "1273-1289"
COMMENT     Sequence Label: L2373-FRB-GPA-20-114
FEATURES             Location/Qualifiers
     source          1..6424
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1..18)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7..231
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      277..705
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(364..383)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     574..595
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(714..734)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        719..1048
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      899..1034
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     961..980
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1097..2122
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1097..2122
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    2252..2373
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2289..2308)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2343..2362
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2422..2438)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2422..2438)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2435..2457)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2446..2462
                     /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(2470..2476,2477..2494,2495..2500))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2515..2536
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2653..2670)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2824..3412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2904..2923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3583..4374,4375..4443))
                     /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     4206..4225
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4444..4548)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4623..4642)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4814..5193
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5194..5397
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5348..5368
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5442..5461
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5442..5460
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5559..5588
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
ORIGIN
        1 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
       61 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
      121 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
      181 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
      241 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
      301 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
      361 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      421 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      481 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      541 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      601 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
      661 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
      721 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
      781 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
      841 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
      901 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
      961 tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg
     1021 cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg
     1081 atctgatcag cacgtgatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     1141 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
     1201 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     1261 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     1321 ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc
     1381 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     1441 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     1501 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     1561 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     1621 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     1681 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     1741 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     1801 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     1861 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     1921 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     1981 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     2041 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     2101 tcgtccgagg gcaaaggaat agcacgtgct acgagatttc gattccaccg ccgccttcta
     2161 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     2221 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     2281 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2341 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     2401 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     2461 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     2521 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2581 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2641 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2701 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2761 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2821 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2881 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2941 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3001 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3061 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3121 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3181 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3241 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3301 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3361 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3421 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3481 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3541 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3601 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3661 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3721 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3781 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3841 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3901 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3961 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4021 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4081 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4141 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4201 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4261 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4321 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4381 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4441 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4501 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4561 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     4621 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     4681 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
     4741 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
     4801 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     4861 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     4921 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     4981 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     5041 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     5101 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     5161 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     5221 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     5281 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     5341 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
     5401 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
     5461 gagacccaag ctggctagcg tttaaactta agcttggtac catgtatgga aaaataatct
     5521 ttgtattact attgtcagaa attgtgagca tatcagcaga gcagaaactc atctcagaag
     5581 aggatctggg cgcgcctatg ctcgagatgt ggcatgaagg cctggaagag gcatctcgtt
     5641 tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta
     5701 tgatggaacg gggcccccag actctgaagg aaacatcctt taatcaggcc tatggtcgag
     5761 atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc
     5821 tcctccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag actagaaccg
     5881 gtttaagtac cactgaggtg gcaatgcaca cttcaacctc ttcttcagtc acaaagagtt
     5941 acatctcatc acagacaaat gatacgcaca aacgggacac atatgcagcc actcctagag
     6001 ctcatgaagt ttcagaaatt tctgttagaa ctgtttaccc tccagaagag gaaaccggag
     6061 aaagggtaca acttgcccat catttctctg aaccagagat aacactcatt atttttgggg
     6121 tgatggctgg tgttattgga acgatcctct taatttctta cggtattcgc cgactgataa
     6181 agaaaagccc atctgatgta aaacctctcc cctcacctga cacagacgtg cctttaagtt
     6241 ctgttgaaat agaaaatcca gagacaagtg atcaattaat taaggggggg tctggaggca
     6301 gtggtgggtc tggtggtggt tctgggggga gtgggggttc aggcgtgacc ggctaccggc
     6361 tgttcgagga gattctgtaa gcggccgctc gagtctagag ggcccgttta aacccgctga
     6421 tcag
//
