LOCUS       sequence-328828-        7610 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Rap1A biosensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7610)
  AUTHORS   O'Shaughnessy EC, Stone OJ, LaFosse PK, Azoitei ML, Tsygankov D, 
            Heddleston JM, Legant WR, Wittchen ES, Burridge K, Elston TC, Betzig
            E, Chew TL, Adalsteinsson D, Hahn KM
  TITLE     Software for lattice light-sheet imaging of FRET biosensors, 
            illustrated with a new Rap1 biosensor.
  JOURNAL   J Cell Biol. 2019 Sep 2;218(9):3153-3160. doi: 
            10.1083/jcb.201903019. Epub 2019 Aug 23.
  PUBMED    31444239
REFERENCE   2  (bases 1 to 7610)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1083/jcb.201903019"; journalName: "J Cell Biol"; date: 
            "2019-09-2- 2"; volume: "218"; issue: "9"; pages: "3153-3160"
COMMENT     Sequence Label: Rap1A FLARE
FEATURES             Location/Qualifiers
     source          1..7610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1076..1379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1381..1583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1534..1554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1786..1808
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1870..1889
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1931..1950
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1931..1949
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1950..1974
                     /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        1990..2099
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             2117..2146
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3173..3202
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3218..3274
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3332..3385
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3392..3415
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(3425..3427,3428..3430,3431..4141)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     4078..4099
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4753..4785
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             4792..4815
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4905..4924)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4970..5025
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5024..5043)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5113..5160
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5173..5878
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5261..5633
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5290..5878)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5887..5920)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(6088..6675)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6167..6186)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6688..7479,7480..7548))
                     /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     7311..7330
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggcct
     1021 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1081 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1141 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1201 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc
     1261 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1321 ccttacggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc
     1381 tgatgcggtt ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc
     1441 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1501 ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt
     1561 gggaggtcta tataagcaga cgtcgtttag tgaaccgtca gatcactaga tgctttattg
     1621 cggtagttta tcacagttaa attgctaacg ccagtctcga acttaacgtg cagaagttgg
     1681 tcgtgaggca ctgggcaggt aagtatcggg ccctttgtgc ggggggagcg gctcggggct
     1741 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1801 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     1861 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc
     1921 ggaccgaaat taatacgact cactataggg gaattgtgag cggataacaa ttccccggag
     1981 ttaatccggg acctttaatt caacccaaca caatatatta tagttaaata agaattatta
     2041 tcaaatcatt tgtatattaa ttaaaatact atactgtaaa ttacatttta tttacaatca
     2101 aaggagatat accatggagc agaagctgat ctccgaggag gacctgggat ctggatctgc
     2161 tagcatggac tgctgtatca tccgcgtcag cctggacgtg gacaatggca acatgtacaa
     2221 gagcatcctg gtgaccagcc aagataaggc tccggctgta atccgcaagg ccatggacaa
     2281 acacaacctg gaggaggagg agccggagga ctatgagctg ctgcagattc tctcagatga
     2341 ccggaagctg aagatccctg aaaacgccaa cgtcttctat gccatgaact ctaccgccaa
     2401 ctatgacttt gtcctcaaga agcggacctt ctccggagga tccggttcac tcgagatggt
     2461 gagcaagggc gaagagctgt tcaccggcgt ggtgcccatc ctggtggagc tggacggcga
     2521 cgtgaacggc cacaagttca gcgtgagcgg cgagggcgag ggcgacgcca cctacggcaa
     2581 gctgaccctg aagctgctgt gcaccaccgg caagctgccc gtgccctggc ccaccctggt
     2641 gaccaccctg ggctacggcg tgcagtgctt cgcccggtac cccgaccaca tgaagcagca
     2701 cgacttcttc aagagcgcca tgcccgaggg ctacgtgcag gagcggacca tcttcttcaa
     2761 ggacgacggc aactacaaga cccgggccga ggtgaagttc gagggcgaca ccctggtgaa
     2821 ccggatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg gccacaagct
     2881 ggagtacaac tacaacagcc acaacgtgta catcaccgcc gacaagcaga agaacggcat
     2941 caaggccaac ttcaagatcc ggcacaacat cgaggacggc ggcgtgcagc tggccgacca
     3001 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     3061 gagctaccag agcgccctgt tcaaggaccc caacgagaag cgggaccaca tggtgctgct
     3121 ggagttcctg accgccgccg gcatcaccga gggcatggac gagctgtaca aggagcagaa
     3181 gctgatctcc gaggaggacc tggaattcgg aagcggagct actaacttca gcctgctgaa
     3241 gcaggctgga gacgtggagg agaaccctgg acctctgcag aggcctaact cgaagaagac
     3301 atccaacagc cagaaaatta atggcagtgg agagggcaga ggaagtctgc taacatgcgg
     3361 tgacgtcgag gagaatcctg gcccactgca ggactataag gacgatgatg acaaaggtac
     3421 cggtatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     3481 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     3541 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     3601 caccctcgtg accaccctgt cctggggcgt gcagtgcttc gcccgctacc ccgaccacat
     3661 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     3721 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     3781 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     3841 gcacaagctg gagtacaacg ccatccacgg caacgtctat atcaccgccg acaagcagaa
     3901 gaacggcatc aaggccaact tcggcctcaa ctgcaacatc gaggacggca gcgtgcagct
     3961 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     4021 ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat
     4081 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     4141 gtccggactc agatctcgag ctaagcttat gcgtgagtac aagctagtgg tccttggttc
     4201 aggaggcgtt gggaagtctg ctctgacagt tcagtttgtt cagggaattt ttgttgaaaa
     4261 atatgaccca acgatagaag attcctacag aaagcaagtt gaagtcgatt gccaacagtg
     4321 tatgctcgaa atcctggata ctgcagggac agagcaattt acagcaatga gggatttgta
     4381 tatgaagaac ggccaaggtt ttgcactagt atattctatt acagctcagt ccacgtttaa
     4441 cgacttacag gacctgaggg aacagatttt acgggttaag gacacggaag atgttccaat
     4501 gattttggtt ggcaataaat gtgacctgga agatgagcga gtagttggca aagagcaggg
     4561 ccagaattta gcaagacagt ggtgtaactg tgccttttta gaatcttctg caaagtcaaa
     4621 gatcaatgtt aatgagatat tttatgacct ggtcagacag ataaatagga aaacaccagt
     4681 ggaaaagaag aagcctaaaa agaaatcatg tctgctgctc tagcggccgc acagctgtat
     4741 acacgtgcaa gccagccaga actcgccccg gaagaccccg aggatctcga gcaccaccat
     4801 caccatcacc atcactaagt gattaacctc aggtgcaggc tgcctatcag aaggtggtgg
     4861 ctggtgtggc caatgccctg gctcacaaat accactgaga tcgatctttt tccctctgcc
     4921 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     4981 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     5041 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     5101 gcccatatgt aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     5161 ctgaaagcat gcggaggaaa ttctccttga agtttccctg gtgttcaaag taaaggagtt
     5221 tgcaccagac gcacctctgt tcactggtcc ggcgtattaa aacacgatac attgttatta
     5281 gtacatttat taagcgctag attctgtgcg ttgttgattt acagacaatt gttgtacgta
     5341 ttttaataat tcattaaatt tataatcttt agggtggtat gttagagcga aaatcaaatg
     5401 attttcagcg tctttatatc tgaatttaaa tattaaatcc tcaatagatt tgtaaaatag
     5461 gtttcgatta gtttcaaaca agggttgttt ttccgaaccg atggctggac tatctaatgg
     5521 attttcgctc aacgccacaa aacttgccaa atcttgtagc agcaatctag ctttgtcgat
     5581 attcgtttgt gttttgtttt gtaataaagg ttcgacgtcg ttcaaaatat tatgcgcttt
     5641 tgtatttctt tcatcactgt cgttagtgta caattgactc gacgtaaaca cgttaaatag
     5701 agcttggaca tatttaacat cgggcgtgtt agctttatta ggccgattat cgtcgtcgtc
     5761 ccaaccctcg tcgttagaag ttgcttccga agacgatttt gccatagcca cacgacgcct
     5821 attaattgtg tcggctaaca cgtccgcgat caaatttgta gttgagcttt ttggaattgc
     5881 gatcgcataa cttcgtatag catacattat acgaagttat aagctcggaa cgctgcgctc
     5941 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     6001 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     6061 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgccccctga cgagcatcac
     6121 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6181 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6241 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6301 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6361 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6421 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6481 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6541 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6601 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6661 aaaaaaggat ctcaagaaga tcctttgtta ccaatgctta atcagtgagg cacctatctc
     6721 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6781 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6841 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6901 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6961 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7021 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7081 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7141 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7201 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7261 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7321 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7381 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7441 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7501 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7561 tcaatattat tgaagcattt atcagggtta ttgtctcatg tccgcgcgtt
//
