LOCUS       sequence-174912-        6606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  rapamycin-mediated heterodimerization between PCRD and DCRD from 
            C-terminal of L-type Ca2+ channels.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6606)
  AUTHORS   Liu N, Yang Y, Ge L, Liu M, Colecraft HM, Liu X
  TITLE     Cooperative and acute inhibitionby multiple C-terminal motifs of 
            L-type Ca2+ channels.
  JOURNAL   Elife. 2017 Jan 6;6. pii: e21989. doi: 10.7554/eLife.21989.
  PUBMED    28059704
REFERENCE   2  (bases 1 to 6606)
  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"; journalName: "Elife."; 
            date: "2017-01-6"; volume: "6. pii: e21989. doi"; pages: " 
            10.7554/eLife.21989"
COMMENT     Sequence Label: CFP-PCRD-FRB
FEATURES             Location/Qualifiers
     source          1..6606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    683..845
                     /label=QBI SP163
                     /note="strong translational enhancer derived from the 5' 
                     UTR of the mouse VEGF gene"
     CDS             858..875
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             879..911
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(915..923,924..938)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             join(945..947,948..950,951..1661)
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(990..1011)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1251..1270)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1598..1619
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2061..2339
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     primer_bind     complement(2380..2397)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2386..2610
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2656..3084
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2743..2762)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2953..2974
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3093..3113)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3098..3427
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3278..3413
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3340..3359
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3475..3522
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3541..3915
                     /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    4045..4166
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4082..4101)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4136..4155
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4215..4231)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4215..4231)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4228..4250)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4239..4255
                     /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(4263..4269,4270..4287,4288..4293))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4308..4329
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4446..4463)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4617..5205)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4697..4716)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5376..6167,6168..6236))
                     /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     5999..6018
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6237..6341)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6416..6435)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacttaagc ttagcgcaga ggcttggggc agccgagcgg cagccaggcc
      721 ccggcccggg cctcggttcc agaagggaga ggagcccgcc aaggcgcgca agagagcggg
      781 ctgcctcgca gtccgagccg gagagggagc gcgagccgcg ccggccccgg acggcctccg
      841 aaaccatggg gggttctcat catcatcatc atcatggtat ggctagcatg actggtggac
      901 agcaaatggg tcgggatctg tacgacgatg acgataaggt accaatggtg agcaagggcg
      961 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1021 acaggttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1081 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1141 cctggggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1201 agtccgccat gcccgaaggc tacgtccagg agcgtaccat cttcttcaag gacgacggca
     1261 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1321 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1381 acatcagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc aaggcccact
     1441 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1501 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     1561 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1621 ccgccgccgg gatcactctc ggcatggacg agctgtacaa ggcggccgct aacgacgccg
     1681 cccctagcac ctcttccgcc cttcaggctg gtctgcggag cctgcaggac ttgggtcctg
     1741 agatgcggca ggccctcacc tgtgacacag aggaggagga agaagagggg caggagggag
     1801 tggaggagga agatgaaaag gacttggaaa ctaacaaagc cacgatggtc tcccagccct
     1861 cagctcgccg gggctccggg atttctgtgt ctctgcctgt cggggacaga cttccagatt
     1921 cactctcctt tgggcccagt gatgatgaca gggggactcc cacctccagt cagcccagtg
     1981 tgccccaggc tggatccaac acccacagga gaggctctgg ggctctcatt ttcaccatcc
     2041 cagaagaagg aaatgaattc atcctctggc atgagatgtg gcatgaaggc ctggaagagg
     2101 catctcgttt gtactttggg gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct
     2161 tgcatgctat gatggaacgg ggcccccaga ctctgaagga aacatccttt aatcaggcct
     2221 atggtcgaga tttaatggag gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg
     2281 tcaaggacct cctccaagcc tgggacctct attatcatgt gttccgacga atctcaaagt
     2341 gactcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     2401 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2461 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2521 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2581 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2641 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2701 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2761 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2821 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2881 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2941 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3001 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3061 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     3121 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3181 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3241 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3301 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3361 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3421 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac
     3481 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc
     3541 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc
     3601 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt
     3661 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac
     3721 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag
     3781 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag
     3841 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc
     3901 gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3961 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4021 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4081 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4141 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4201 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4261 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4321 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4381 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4441 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4501 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4561 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4621 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4681 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4741 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4801 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4861 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4921 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4981 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5041 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5101 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5161 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5221 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5281 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5341 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5401 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5461 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5521 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5581 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5641 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5701 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5761 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5821 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5881 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5941 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6001 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6061 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6121 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6181 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6241 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6301 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6361 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     6421 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     6481 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     6541 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     6601 cgcgtt
//
