LOCUS       sequence-468025-        5555 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of RspA-CFAST on the ER membrane.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5555)
  AUTHORS   Garcia Casas P, Rossini M, Pavenius L, Saeed M, Arnst N, Sonda S, 
            Fernandes T, D'Arsie I, Bruzzone M, Berno V, Raimondi A, Sassano ML,
            Naia L, Barbieri E, Sigismund S, Agostinis P, Sturlese M, Niemeyer 
            BA, Brismar H, Ankarcrona M, Gautier A, Pizzo P, Filadi R
  TITLE     Simultaneous detection of membrane contact dynamics and associated 
            Ca(2+) signals by reversible chemogenetic reporters.
  JOURNAL   Nat Commun. 2024 Nov 12;15(1):9775. doi: 10.1038/s41467-024-52985-0.
  PUBMED    39532847
REFERENCE   2  (bases 1 to 5555)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-11-12"; volume: "15(1):9775. doi"; pages: " 
            10.1038/s41467-024-52985-0"
COMMENT     Sequence Label: ER-RspA-CFAST
FEATURES             Location/Qualifiers
     source          1..5555
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      895..904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1108..1126)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1109..1126)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1146..1163)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1152..1376
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1422..1850
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1509..1528)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1719..1740
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1859..1879)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1864..2193
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2044..2179
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2106..2125
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2260..3054
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2314..2333)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2924..2943
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3228..3349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3265..3284)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3319..3338
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3398..3414)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3398..3414)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3411..3433)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3422..3438
                     /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(3446..3452,3453..3470,3471..3476))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3491..3512
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3629..3646)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3800..4388)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3880..3899)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4559..5350,5351..5419))
                     /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     5182..5201
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5420..5524)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgccacc
      901 atggaccctg tggtggtgct ggggctctgt ctctcctgtt tgcttctcct ttcactctgg
      961 aaacagagct atgggggagg gtctggtgga tctggaggta ccgatatcgt ctccggagga
     1021 ggcggcagcg gcggaggggg atccggcgac agcttctgga tcttcgtgaa gagactgtaa
     1081 ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg
     1141 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     1201 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     1261 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     1321 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     1381 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     1441 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1501 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1561 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1621 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1681 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1741 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1801 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     1861 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     1921 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     1981 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2041 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2101 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     2161 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     2221 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     2281 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     2341 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     2401 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     2461 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     2521 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     2581 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     2641 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     2701 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     2761 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     2821 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     2881 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     2941 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     3001 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     3061 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3121 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3181 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     3241 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3301 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3361 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3421 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3481 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3541 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3601 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3661 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3721 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3781 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3841 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3901 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3961 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4021 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4081 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4141 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4201 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4261 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4321 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4381 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4441 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4501 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4561 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4621 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4681 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4741 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4801 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4861 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4921 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4981 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5041 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5101 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5161 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5221 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5281 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5341 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5401 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5461 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5521 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
