LOCUS       sequence-223493-        5500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encodes a membrane located inhibitor of Ga12/13 signaling 
            (RGS domain from p115).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5500)
  AUTHORS   Reinhard NR, Mastop M, Yin T, Wu Y, Bosma EK, Gadella TWJ Jr, 
            Goedhart J, Hordijk PL
  TITLE     The balance between Galphai-Cdc42/Rac and Galpha12/13-RhoA pathways 
            determines endothelial barrier regulation by 
            sphingosine-1-phosphate.
  JOURNAL   Mol Biol Cell. 2017 Nov 7;28(23):3371-3382. doi: 
            10.1091/mbc.E17-03-0136. Epub 2017 Sep 27.
  PUBMED    28954861
REFERENCE   2  (bases 1 to 5500)
  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: 
            "10.1091/mbc.E17-03-0136"; journalName: "Mol Biol Cell"; date: 
            "2017-11-7- 7"; volume: "28"; issue: "23"; pages: "3371-3382"
COMMENT     Sequence Label: Lck-mCherry-RGS
FEATURES             Location/Qualifiers
     source          1..5500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      595..604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      640..649
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             646..1353
                     /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(791..809)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1059..1078
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2288..2409
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2325..2344)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2379..2398
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2416..2871)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2553..2574)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2765..2784
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2898..3002
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3004..3361
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3004..3195
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3027..3047)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3212..3347
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3396..4190
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3396..4190
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3450..3469)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4060..4079
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4378..4397)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4422..4469
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4798..5386
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5287..5306
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagccacc
      601 atgggctgcg tgtgcagcag caaccccgag ctaccggtcg ccaccatggt gagcaagggc
      661 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
      721 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
      781 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
      841 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
      901 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
      961 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     1021 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     1081 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     1141 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     1201 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     1261 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     1321 cactccaccg gcggcatgga cgagctgtac aagtccggac tcagatctat ggaagacttc
     1381 gcccgagggg cggcctcccc aggcccctcc cggcctggcc tggttcccgt cagcatcatc
     1441 ggggctgagg atgaggattt tgagaacgag ctggagacaa actcagaaga gcaaaacagc
     1501 cagttccaga gcctggagca ggtgaagcgg cgcccagccc acctcatggc cctcctgcag
     1561 cacgtggccc tgcagtttga gccaggaccc ctgctttgct gtctgcatgc cgacatgctg
     1621 ggctcactgg gccccaagga ggccaagaag gccttcctgg acttctacca cagcttcctg
     1681 gagaagacag cggttctccg ggtgccggtc cctcccaacg tcgcctttga acttgaccgc
     1741 actagggctg acctcatctc cgaggatgtc cagcggcggt tcgtgcagga ggtggtgcaa
     1801 agccagcagg tagccgtggg ccggcagctg gaggacttcc gttccaagcg gctcatgggc
     1861 atgacgccct gggagcagga gctggcccag ctggaggctt gggttgggcg ggaccgagcc
     1921 agctacgagg cccgggagcg gcacgtggcg gagcggctgc tcatgcacct ggaggagatg
     1981 caacatacca tctctaccga cgaagaaaag agtgctgccg tggtcaacgc cattggcctg
     2041 tacatgcgcc accttggggt gcggaccaag agtggagaca agaagtcggg gaggaacttc
     2101 ttccggaaaa aggtgatggg gaactaagaa ttctgcagtc gacggtaccg cgggcccggg
     2161 atccaccgga tctagataac tgatcataat cagccatacc acatttgtag aggttttact
     2221 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2281 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2341 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2401 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     2461 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     2521 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2581 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2641 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2701 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2761 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2821 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     2881 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2941 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3001 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3061 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3121 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3181 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3241 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3301 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3361 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3421 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3481 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3541 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     3601 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3661 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3721 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3781 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3841 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     3901 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     3961 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4021 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4081 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4141 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4201 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4261 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4321 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga
     4381 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     4441 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca
     4501 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc
     4561 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc
     4621 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt
     4681 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4741 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4801 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4861 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     4921 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     4981 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5041 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5101 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5161 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5221 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5281 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5341 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5401 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5461 atcccctgat tctgtggata accgtattac cgccatgcat
//
