LOCUS       sequence-195040-        8550 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Rock1-GFP for the study of localisation 
            signaling..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8550)
  AUTHORS   Priya R, Gomez GA, Budnar S, Verma S, Cox HL, Hamilton NA, Yap AS
  TITLE     Feedback regulation through myosin II confers robustness on RhoA 
            signalling at E-cadherin junctions.
  JOURNAL   Nat Cell Biol. 2015 Oct;17(10):1282-93. doi: 10.1038/ncb3239. Epub 
            2015 Sep 14.
  PUBMED    26368311
REFERENCE   2  (bases 1 to 8550)
  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: "10.1038/ncb3239"; 
            journalName: "Nat Cell Biol"; date: "2015-10"; volume: "17"; issue: 
            "10"; pages: "1282-93"
COMMENT     Sequence Label: GFP-mROCK1-delta4
FEATURES             Location/Qualifiers
     source          1..8550
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      26..614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     515..534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        789..1092
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1093..1296
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1247..1267
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1335..1344
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1341..1343,1344..1346,1347..2057)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1341..1343,1344..1346,1347..2057)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1386..1407)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1647..1666)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1994..2015
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    6066..6187
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6103..6122)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6157..6176
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(6194..6649)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6331..6352)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6543..6562
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6676..6780
                     /gene="bla"
                     /label=AmpR promoter
     promoter        6782..7139
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6782..6973
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6805..6825)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6990..7125
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             7174..7968
                     /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             7174..7968
                     /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(7228..7247)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7838..7857
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(8156..8175)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    8200..8247
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
       61 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      121 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      181 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      241 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      301 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      361 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      421 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      481 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      541 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      601 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      661 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      721 ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      781 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      841 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      901 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      961 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     1021 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     1081 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     1141 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     1201 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     1261 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc
     1321 tagcgctacc ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     1381 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     1441 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1501 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1561 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1621 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1681 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1741 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     1801 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     1861 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1921 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     1981 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2041 tggacgagct gtacaagtcc ggactcagat ctcgagctca atatgaagtg gtaaaggtaa
     2101 tcggcagagg tgcatttgga gaagttcaat tggtgaggca taaatccact aggaaggttt
     2161 atgctatgaa gctgctcagc aaatttgaaa tgattaagag atcggattct gcttttttct
     2221 gggaagaaag ggacatcatg gcgtttgcca atagtccttg ggttgttcag cttttttatg
     2281 cattccaaga tgaccggtat ctctacatgg tgatggagta catgcctggc ggagaccttg
     2341 tcaacctgat gagcaactat gacgtgcctg aaaaatgggc aagattctat actgcagaag
     2401 tagttcttgc attggatgca atccattcca tgggttttat tcatagagat gtaaagcctg
     2461 ataacatgct gctggataag tctggacatt tgaagttagc agattttggt acttgtatga
     2521 agatgaataa ggaaggtatg gtacgatgtg acacagcagt tggaacacct gattatatct
     2581 cacccgaagt attaaaatcc caaggcggtg atggctatta tggacgagag tgtgactggt
     2641 ggtcagttgg ggtatttcta tatgaaatgc tcgtaggtga cacacctttc tatgcagatt
     2701 ctttggttgg gacttacagt aaaattatga accataaaaa ttcacttact ttccctgatg
     2761 ataatgacat atcaaaagag gcaaaaaatc tcatttgtgc cttccttaca gacagagaag
     2821 tgagattggg cagaaatggt gtagaggaaa tcaaacgtca tctcttcttc aaaaatgatc
     2881 agtgggcttg ggaaacgctc cgagacactg tagcaccagt tgtgcctgat ttaagtagtg
     2941 acattgatac tagtaatttt gatgacttag aagaagataa aggagatgaa gaaacatttc
     3001 ctattccaaa agcttttgtt ggcaatcagc taccttttgt aggatttaca tattatagca
     3061 atcgtagata cttaccttca gcaaatgcca gtgagaacag atctagctcc aatgtggata
     3121 aaagcttgca ggaaagttta cagaaaacaa tctacaagct tgaagagcaa ctgcataatg
     3181 aaatgcaatt aaaagatgaa atggagcaga aatgcagaac ttcaaactta aaactagaca
     3241 agataatgaa agaattggat gaagagggga atcaaagaag aaatctagaa tctgcagtgt
     3301 ctcaaattga gaaagaaaag atgttgctac aacatagaat taatgagtac caaagaaaag
     3361 tggaacagga aaatgagaag agaagaaata tagaaaatga agtttctaca ctaaaggacc
     3421 agttagaaga cttacggaaa gcaagtcaga cctcacagct tgctaatgag aagctgacac
     3481 agttacagaa gcagttagaa gaagcgaatg acttacttag gacagaatca gacacagctg
     3541 taagattaag gaagagtcac acagagatga gcaagtcaat tagtcagcta gagtccctga
     3601 acagagaatt acaggaaaga aacagaattt tagagaattc caagtcacaa gcagacaagg
     3661 attactacca gctgcaagct gtattggaag ctgagcgaag agacagaggg catgattctg
     3721 agatgattgg agaccttcaa gcacgaatta catctttaca agaagaagtg aaacatctca
     3781 aacataatct tgaaagagtg gaaggagaaa gaaaagaggc tcaagacatg cttaatcatt
     3841 cagaaaagga aaagaataat ttggagatag atttgaatta caagctcaag tctatacaac
     3901 agcggttaga acaagaagta aatgaacata aagtcaccaa agcacgccta actgacaagc
     3961 accaatctat cgaagaggca aagtctgttg ccatgtgtga aatggaaaaa aagctgaagg
     4021 aagagagaga agctcgagag aaggcagaaa atagagttgt tgagactgag aaacaatgtt
     4081 ccatgctaga tgttgacctg aaacagtctc agcagaagct agaacatttg accgaaaata
     4141 aagaaaggat ggaggatgaa gttaagaatc tcgccctgca gctggaacag gaatcaaata
     4201 aacggctctt gttacagaat gaattgaaga ctcaagcatt tgaagcagac aatttaaaag
     4261 gtttagaaaa acagatgaaa caggaaataa atactttatt ggaagcaaag agattgttag
     4321 aatttgagtt agctcagctt acaaagcaat ataggggaaa tgaagggcaa atgcgggagt
     4381 tacaagatca acttgaagct gagcagtatt tctctacact ttataaaacc caagtaaagg
     4441 agcttaaaga agaaattgaa gaaaaaaacc gagaaaattt aaggaaaata caggaactgc
     4501 aaagtgaaaa agaaactctc tctactcagt tggatttagc agaaacaaaa gctgagtctg
     4561 agcagctggc gcgagggatt ctggaagaac agtattttga gttgactcag gaaagcaaga
     4621 aagctgcttc aagaaataga caagagatta cagataaaga tcatactgtt agtcggcttg
     4681 aagaaacaaa cagtgtgcta accaaagata ttgaaatgct aagaaaagaa aatgaagagc
     4741 taaatgaaag aatgaggaca gcagaagaag aatataaatt gaagaaggag gaagaaataa
     4801 ataatcttaa ggctgctttt gaaaagaata tcagtaccga acggaccctt aaaacacagg
     4861 ctgttaacaa gttggcagaa ataatgaatc gcaaagattt taaaattgat cgaaagaaag
     4921 ctaatacaca agatttaaga aaaaaagaaa aggaaaatag aaagctacaa ctggaactta
     4981 accaagagag agaaaaattt aaccaaatgg tggtgaaaca ccagaaggag ctgaatgaca
     5041 tgcaagcgca attggtagaa gaatgtacac atagaaatga acttcaaatg cagttggcta
     5101 gcaaagagag tgatattgag cagttgcggg caaaactttt ggacctttcg gattcaacta
     5161 gtgttgctag ttttcctagt gcagatgaaa ctgatggaaa ccttccagag tcaagaattg
     5221 aaggttggct ttctgtacca aatagaggaa atatcaaaag atatggctgg aagaaacagt
     5281 atgttgtggt aagcagcaaa aagatcttgt tctataatga tgaacaagat aaggaacaat
     5341 ccagtccatc tatggtgtta gacatagata aattgtttca tgtccgacct gtaacccaag
     5401 gagatgtgta cagagcagaa actgaagaaa ttcctaaaat attccagata ctatatgcaa
     5461 atgaaggtga atgtagaaaa gatatagaag tggaaccggt tcaacaaggt gaaaaaacta
     5521 atttccagaa tcacaaaggc catgaattca ttcctaccct ctaccacttt cctgccaact
     5581 gtgaggcctg tgccaaacct ttgtggcatg tctttaagcc accacctgcc ctagagtgtc
     5641 gaagatgcca tgttaagtgc cacagagacc atctagacaa gaaagaggac ttgatttccc
     5701 cgtgcaaagt aagttacgat gtaacatcag caagagatat gctgctgtta gcatgttctc
     5761 aggatgaaca aaaaaagtgg gttactcatc tagtaaagaa gattcctaag aacccaccat
     5821 ctggctttgt ccgtgcctct cctcgaacgc tgtcaacaag atccactgca aatcagtctt
     5881 tccgcaaagt agtcaaaaat acatctggaa aaactagtta aggtaccgcg ggcccgggat
     5941 ccaccggatc tagataactg atcataatca gccataccac atttgtagag gttttacttg
     6001 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     6061 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     6121 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     6181 tatcttaacg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     6241 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     6301 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     6361 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     6421 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     6481 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     6541 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     6601 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     6661 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     6721 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     6781 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     6841 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     6901 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     6961 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     7021 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     7081 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     7141 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     7201 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     7261 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     7321 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     7381 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     7441 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     7501 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     7561 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     7621 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     7681 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     7741 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     7801 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     7861 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     7921 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     7981 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     8041 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     8101 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     8161 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     8221 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     8281 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     8341 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     8401 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     8461 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     8521 gtgagttttc gttccactga gcgtcagacc
//
