LOCUS       sequence-195049-        6597 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 6597)
  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 6597)
  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-delta7
FEATURES             Location/Qualifiers
     source          1..6597
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      20..608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     509..528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        783..1086
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1087..1290
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1241..1261
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1329..1338
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1335..1337,1338..1340,1341..2051)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1335..1337,1338..1340,1341..2051)
                     /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(1380..1401)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1641..1660)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1988..2009
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4107..4228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4144..4163)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4198..4217
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4235..4690)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4372..4393)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4584..4603
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4717..4821
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4823..5180
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4823..5014
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4846..4866)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5031..5166
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5215..6009
                     /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             5215..6009
                     /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(5269..5288)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5879..5898
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6197..6216)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6241..6288
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
       61 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
      121 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
      181 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      241 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      301 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
      361 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      421 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      481 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      541 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      601 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      661 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc
      721 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      781 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      841 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      901 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      961 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     1021 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     1081 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     1141 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     1201 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     1261 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
     1321 taccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1381 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     1441 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1501 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     1561 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     1621 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1681 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1741 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1801 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1861 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1921 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     1981 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     2041 agctgtacaa gtccggactc agatctcgag ctcaacgaga gaaggcagaa aatagagttg
     2101 ttgagactga gaaacaatgt tccatgctag atgttgacct gaaacagtct cagcagaagc
     2161 tagaacattt gaccgaaaat aaagaaagga tggaggatga agttaagaat ctcgccctgc
     2221 agctggaaca ggaatcaaat aaacggctct tgttacagaa tgaattgaag actcaagcat
     2281 ttgaagcaga caatttaaaa ggtttagaaa aacagatgaa acaggaaata aatactttat
     2341 tggaagcaaa gagattgtta gaatttgagt tagctcagct tacaaagcaa tataggggaa
     2401 atgaagggca aatgcgggag ttacaagatc aacttgaagc tgagcagtat ttctctacac
     2461 tttataaaac ccaagtaaag gagcttaaag aagaaattga agaaaaaaac cgagaaaatt
     2521 taaggaaaat acaggaactg caaagtgaaa aagaaactct ctctactcag ttggatttag
     2581 cagaaacaaa agctgagtct gagcagctgg cgcgagggat tctggaagaa cagtattttg
     2641 agttgactca ggaaagcaag aaagctgctt caagaaatag acaagagatt acagataaag
     2701 atcatactgt tagtcggctt gaagaaacaa acagtgtgct aaccaaagat attgaaatgc
     2761 taagaaaaga aaatgaagag ctaaatgaaa gaatgaggac agcagaagaa gaatataaat
     2821 tgaagaagga ggaagaaata aataatctta aggctgcttt tgaaaagaat atcagtaccg
     2881 aacggaccct taaaacacag gctgttaaca agttggcaga aataatgaat cgcaaagatt
     2941 ttaaaattga tcgaaagaaa gctaatacac aagatttaag aaaaaaagaa aaggaaaata
     3001 gaaagctaca actggaactt aaccaagaga gagaaaaatt taaccaaatg gtggtgaaac
     3061 accagaagga gctgaatgac atgcaagcgc aattggtaga agaatgtaca catagaaatg
     3121 aacttcaaat gcagttggct agcaaagaga gtgatattga gcagttgcgg gcaaaacttt
     3181 tggacctttc ggattcaact agtgttgcta gttttcctag tgcagatgaa actgatggaa
     3241 accttccaga gtcaagaatt gaaggttggc tttctgtacc aaatagagga aatatcaaaa
     3301 gatatggctg gaagaaacag tatgttgtgg taagcagcaa aaagatcttg ttctataatg
     3361 atgaacaaga taaggaacaa tccagtccat ctatggtgtt agacatagat aaattgtttc
     3421 atgtccgacc tgtaacccaa ggagatgtgt acagagcaga aactgaagaa attcctaaaa
     3481 tattccagat actatatgca aatgaaggtg aatgtagaaa agatatagaa gtggaaccgg
     3541 ttcaacaagg tgaaaaaact aatttccaga atcacaaagg ccatgaattc attcctaccc
     3601 tctaccactt tcctgccaac tgtgaggcct gtgccaaacc tttgtggcat gtctttaagc
     3661 caccacctgc cctagagtgt cgaagatgcc atgttaagtg ccacagagac catctagaca
     3721 agaaagagga cttgatttcc ccgtgcaaag taagttacga tgtaacatca gcaagagata
     3781 tgctgctgtt agcatgttct caggatgaac aaaaaaagtg ggttactcat ctagtaaaga
     3841 agattcctaa gaacccacca tctggctttg tccgtgcctc tcctcgaacg ctgtcaacaa
     3901 gatccactgc aaatcagtct ttccgcaaag tagtcaaaaa tacatctgga aaaactagtt
     3961 aaggtaccgc gggcccggga tccaccggat ctagataact gatcataatc agccatacca
     4021 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     4081 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     4141 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4201 gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta
     4261 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     4321 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     4381 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     4441 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     4501 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     4561 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     4621 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     4681 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     4741 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     4801 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     4861 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4921 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4981 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     5041 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     5101 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     5161 gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt
     5221 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     5281 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     5341 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     5401 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     5461 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     5521 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     5581 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     5641 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5701 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     5761 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5821 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5881 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5941 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     6001 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     6061 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     6121 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     6181 ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     6241 cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg
     6301 ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg
     6361 cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg
     6421 cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga
     6481 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6541 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtag
//
