LOCUS       sequence-195027-        5838 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 5838)
  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 5838)
  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-delta1
FEATURES             Location/Qualifiers
     source          1..5838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      18..606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     507..526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        781..1084
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1085..1288
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1239..1259
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1327..1336
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1333..1335,1336..1338,1339..2049)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1333..1335,1336..1338,1339..2049)
                     /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(1378..1399)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1639..1658)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1986..2007
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3346..3467
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3383..3402)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3437..3456
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3474..3929)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3611..3632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3823..3842
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3956..4060
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4062..4419
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4062..4253
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4085..4105)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4270..4405
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4454..5248
                     /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             4454..5248
                     /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(4508..4527)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5118..5137
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5436..5455)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5480..5527
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
       61 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
      121 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
      181 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
      241 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
      301 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
      361 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
      421 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
      481 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
      541 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
      601 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
      661 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
      721 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      781 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      841 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      901 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      961 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
     1021 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
     1081 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
     1141 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     1201 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     1261 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
     1321 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1381 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1441 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1501 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1561 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1621 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1681 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1741 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1801 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1861 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1921 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1981 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     2041 ctgtacaagt ccggactcag atctcgagct caaatgtcga ctggggacag ttttgagact
     2101 cggtttgaaa aaatcgacaa cctgctgcgg gatcccaaat cggaagtgaa ttcagattgt
     2161 ttgctggatg gattggatgc cctggtttat gatttggatt ttcctgcctt aagaaaaaac
     2221 aaaaatattg acaactttct aagcagatat aaagacacaa taaataaaat tagagattta
     2281 cggatgaaag cagaagatta tgaagtggta aaggtaatcg gcagaggtgc atttggagaa
     2341 gttcaattgg tgaggcataa atccactagg aaggtttatg ctatgaagct gctcagcaaa
     2401 tttgaaatga ttaagagatc ggattctgct tttttctggg aagaaaggga catcatggcg
     2461 tttgccaata gtccttgggt tgttcagctt ttttatgcat tccaagatga ccggtatctc
     2521 tacatggtga tggagtacat gcctggcgga gaccttgtca acctgatgag caactatgac
     2581 gtgcctgaaa aatgggcaag attctatact gcagaagtag ttcttgcatt ggatgcaatc
     2641 cattccatgg gttttattca tagagatgta aagcctgata acatgctgct ggataagtct
     2701 ggacatttga agttagcaga ttttggtact tgtatgaaga tgaataagga aggtatggta
     2761 cgatgtgaca cagcagttgg aacacctgat tatatctcac ccgaagtatt aaaatcccaa
     2821 ggcggtgatg gctattatgg acgagagtgt gactggtggt cagttggggt atttctatat
     2881 gaaatgctcg taggtgacac acctttctat gcagattctt tggttgggac ttacagtaaa
     2941 attatgaacc ataaaaattc acttactttc cctgatgata atgacatatc aaaagaggca
     3001 aaaaatctca tttgtgcctt ccttacagac agagaagtga gattgggcag aaatggtgta
     3061 gaggaaatca aacgtcatct cttcttcaaa aatgatcagt gggcttggga aacgctccga
     3121 gacactgtag caccagttgt gcctgattta agtagtgaca ttgatactag taattttgat
     3181 gacttagaag aagataaata aggtaccgcg ggcccgggat ccaccggatc tagataactg
     3241 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     3301 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     3361 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3421 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt
     3481 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3541 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     3601 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3661 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3721 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     3781 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     3841 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     3901 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg
     3961 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4021 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag
     4081 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     4141 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     4201 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     4261 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4321 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4381 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg
     4441 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4501 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4561 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4621 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4681 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4741 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4801 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4861 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4921 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4981 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     5041 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     5101 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     5161 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     5221 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     5281 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5341 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5401 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg
     5461 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg
     5521 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     5581 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     5641 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt
     5701 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5761 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5821 gcgtcagacc ccgtagaa
//
