LOCUS       sequence-195035-        6894 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 6894)
  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 6894)
  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-delta2
FEATURES             Location/Qualifiers
     source          1..6894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      14..602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     503..522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        777..1080
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1081..1284
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1235..1255
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1323..1332
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1329..1331,1332..1334,1335..2045)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1329..1331,1332..1334,1335..2045)
                     /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(1374..1395)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1635..1654)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1982..2003
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4398..4519
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4435..4454)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4489..4508
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4526..4981)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4663..4684)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4875..4894
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5008..5112
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5114..5471
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5114..5305
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5137..5157)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5322..5457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5506..6300
                     /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             5506..6300
                     /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(5560..5579)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6170..6189
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6488..6507)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6532..6579
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
       61 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      121 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
      181 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      241 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      301 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      361 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      421 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      481 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      541 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      601 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      661 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcattagt
      721 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      781 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      841 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      901 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      961 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     1021 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     1081 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     1141 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     1201 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     1261 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
     1321 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     1381 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     1441 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     1501 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     1561 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     1621 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     1681 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     1741 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     1801 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     1861 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     1921 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     1981 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     2041 acaagtccgg actcagatct cgagctcaaa tgtcgactgg ggacagtttt gagactcggt
     2101 ttgaaaaaat cgacaacctg ctgcgggatc ccaaatcgga agtgaattca gattgtttgc
     2161 tggatggatt ggatgccctg gtttatgatt tggattttcc tgccttaaga aaaaacaaaa
     2221 atattgacaa ctttctaagc agatataaag acacaataaa taaaattaga gatttacgga
     2281 tgaaagcaga agattatgaa gtggtaaagg taatcggcag aggtgcattt ggagaagttc
     2341 aattggtgag gcataaatcc actaggaagg tttatgctat gaagctgctc agcaaatttg
     2401 aaatgattaa gagatcggat tctgcttttt tctgggaaga aagggacatc atggcgtttg
     2461 ccaatagtcc ttgggttgtt cagctttttt atgcattcca agatgaccgg tatctctaca
     2521 tggtgatgga gtacatgcct ggcggagacc ttgtcaacct gatgagcaac tatgacgtgc
     2581 ctgaaaaatg ggcaagattc tatactgcag aagtagttct tgcattggat gcaatccatt
     2641 ccatgggttt tattcataga gatgtaaagc ctgataacat gctgctggat aagtctggac
     2701 atttgaagtt agcagatttt ggtacttgta tgaagatgaa taaggaaggt atggtacgat
     2761 gtgacacagc agttggaaca cctgattata tctcacccga agtattaaaa tcccaaggcg
     2821 gtgatggcta ttatggacga gagtgtgact ggtggtcagt tggggtattt ctatatgaaa
     2881 tgctcgtagg tgacacacct ttctatgcag attctttggt tgggacttac agtaaaatta
     2941 tgaaccataa aaattcactt actttccctg atgataatga catatcaaaa gaggcaaaaa
     3001 atctcatttg tgccttcctt acagacagag aagtgagatt gggcagaaat ggtgtagagg
     3061 aaatcaaacg tcatctcttc ttcaaaaatg atcagtgggc ttgggaaacg ctccgagaca
     3121 ctgtagcacc agttgtgcct gatttaagta gtgacattga tactagtaat tttgatgact
     3181 tagaagaaga taaaggagat gaagaaacat ttcctattcc aaaagctttt gttggcaatc
     3241 agctaccttt tgtaggattt acatattata gcaatcgtag atacttacct tcagcaaatg
     3301 ccagtgagaa cagatctagc tccaatgtgg ataaaagctt gcaggaaagt ttacagaaaa
     3361 caatctacaa gcttgaagag caactgcata atgaaatgca attaaaagat gaaatggagc
     3421 agaaatgcag aacttcaaac ttaaaactag acaagataat gaaagaattg gatgaagagg
     3481 ggaatcaaag aagaaatcta gaatctgcag tgtctcaaat tgagaaagaa aagatgttgc
     3541 tacaacatag aattaatgag taccaaagaa aagtggaaca ggaaaatgag aagagaagaa
     3601 atatagaaaa tgaagtttct acactaaagg accagttaga agacttacgg aaagcaagtc
     3661 agacctcaca gcttgctaat gagaagctga cacagttaca gaagcagtta gaagaagcga
     3721 atgacttact taggacagaa tcagacacag ctgtaagatt aaggaagagt cacacagaga
     3781 tgagcaagtc aattagtcag ctagagtccc tgaacagaga attacaggaa agaaacagaa
     3841 ttttagagaa ttccaagtca caagcagaca aggattacta ccagctgcaa gctgtattgg
     3901 aagctgagcg aagagacaga gggcatgatt ctgagatgat tggagacctt caagcacgaa
     3961 ttacatcttt acaagaagaa gtgaaacatc tcaaacataa tcttgaaaga gtggaaggag
     4021 aaagaaaaga ggctcaagac atgcttaatc attcagaaaa ggaaaagaat aatttggaga
     4081 tagatttgaa ttacaagctc aagtctatac aacagcggtt agaacaagaa gtaaatgaac
     4141 ataaagtcac caaagcacgc ctaactgaca agcaccaatc tatcgaagag gcaaagtctg
     4201 ttgccatgtg tgaaatggaa aaaaagctga aggaagagag agaagctcga taaggtaccg
     4261 cgggcccggg atccaccgga tctagataac tgatcataat cagccatacc acatttgtag
     4321 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     4381 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4441 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4501 aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     4561 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     4621 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     4681 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     4741 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     4801 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     4861 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     4921 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     4981 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5041 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5101 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     5161 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     5221 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     5281 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     5341 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     5401 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     5461 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     5521 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     5581 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     5641 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     5701 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     5761 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     5821 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     5881 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     5941 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     6001 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     6061 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     6121 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     6181 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     6241 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     6301 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     6361 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     6421 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     6481 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     6541 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     6601 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     6661 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     6721 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     6781 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6841 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aaga
//
