LOCUS       sequence-438760-        6630 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Rho inactivation using rapamycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6630)
  AUTHORS   Marshall-Burghardt S, Migueles-Ramirez RA, Lin Q, El Baba N, Saada 
            R, Umar M, Mavalwala K, Hayer A
  TITLE     Excitable Rho dynamics control cell shape and motility by 
            sequentially activating ERM proteins and actomyosin contractility.
  JOURNAL   Sci Adv. 2024 Sep 6;10(36):eadn6858. doi: 10.1126/sciadv.adn6858. 
            Epub 2024 Sep 6.
  PUBMED    39241071
REFERENCE   2  (bases 1 to 6630)
  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"; journalName: "Sci Adv."; 
            date: "2024-09-6"; volume: "10(36):eadn6858. doi"; pages: " 
            10.1126/sciadv.adn6858. Epub 2024 Sep 6"
COMMENT     Sequence Label: pCAG-mCherry-FKBP-GAP(ARHGAP29)
FEATURES             Location/Qualifiers
     source          1..6630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1739..2446
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1884..1902)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2152..2171
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2459..2779
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     primer_bind     complement(3651..3670)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3716..3771
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3770..3789)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4132..4148)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4132..4148)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4145..4167)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4156..4172
                     /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(4180..4186,4187..4204,4205..4210))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4225..4246
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4305..4500
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4351..4486
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4506..4640
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4543..4562)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4597..4616
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4708..4725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4879..5467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4959..4978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5638..6429,6430..6498))
                     /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     6261..6280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6499..6603)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcaccggtat
     1741 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     1801 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     1861 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     1921 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     1981 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     2041 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     2101 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     2161 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     2221 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     2281 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     2341 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     2401 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagagtg ctggtggtgg
     2461 agtgcaggtg gaaaccatct ccccaggaga cgggcgcacc ttccccaagc gcggccagac
     2521 ctgcgtggtg cactacaccg ggatgcttga agatggaaag aaatttgatt cctcccggga
     2581 cagaaacaag ccctttaagt ttatgctagg caagcaggag gtgatccgag gctgggaaga
     2641 aggggttgcc cagatgagtg tgggtcagag agccaaactg actatatctc cagattatgc
     2701 ctatggtgcc actgggcacc caggcatcat cccaccacat gccactctcg tcttcgatgt
     2761 ggagcttcta aaactggaaa gtgctggtgg tagtgctggt ggtagtgctg gtggtagtgc
     2821 tggtggtagt gctggtggtc ctcgaggtgc ggccgcgcga attttatttg gagcagaatt
     2881 cacacaagtt gcaaaaaagg aaccagatgg tatccctttt atactcaaaa tatgtgcctc
     2941 agagattgaa aatagagctt tgtgtctaca gggaatttat cgtgtgtgtg gaaacaaaat
     3001 aaaaactgaa aaattgtgtc aagctttgga aaatggaatg cacttggtag atatttcaga
     3061 atttagttca catgatatct gtgacgtctt gaaattatac cttcgccagc tcccagaacc
     3121 atttatttta tttcgattgt acaaggaatt tatagacctt gcaaaagaga tccaacatgt
     3181 aaatgaagaa caagagacaa aaaagaatag tcttgaagac aaaaaatggc caaatatgtg
     3241 tatagaaata aaccgaattc ttctaaaaag caaagacctt ctaagacaat tgccagcatc
     3301 aaattttaac agtcttcatt tccttatagt acatctaaag cgggtagtag atcatgcaga
     3361 agaaaacaag atgaactcca aaaacttggg ggtgatattt ggaccaagtc tcattaggcc
     3421 aaggcccaca actgctccta tcaccatctc ctcccttgca gagtattcaa atcaagcacg
     3481 cttggtagag tttctcatta cttactcaca gaagatcttc gatgggtccc tacagccaca
     3541 agatgttatg tgtagcatag gttaagcggc cgcactcctc aggtgcaggc tgcctatcag
     3601 aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc
     3661 tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa
     3721 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg
     3781 acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca
     3841 acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat
     3901 atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag
     3961 atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta
     4021 catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct
     4081 cttctcttat ggagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct
     4141 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4201 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4261 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca
     4321 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4381 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4441 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4501 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4561 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4621 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4681 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4741 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4801 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4861 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4921 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4981 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5041 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5101 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5161 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5221 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5281 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5341 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5401 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5461 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5521 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5581 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5641 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5701 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5761 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5821 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5881 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5941 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6001 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6061 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6121 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6181 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6241 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6301 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6361 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6421 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6481 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6541 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6601 gcgcacattt ccccgaaaag tgccacctgg
//
