LOCUS       sequence-371993-        8775 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express GFP ROCK1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8775)
  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 8775)
  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-ROCK1
FEATURES             Location/Qualifiers
     source          1..8775
                     /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    6283..6404
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6320..6339)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6374..6393
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(6411..6866)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6548..6569)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6760..6779
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6893..6997
                     /gene="bla"
                     /label=AmpR promoter
     promoter        6999..7356
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6999..7190
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(7022..7042)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7207..7342
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             7391..8185
                     /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             7391..8185
                     /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(7445..7464)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     8055..8074
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(8373..8392)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    8417..8464
                     /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 aagataaagg agatgaagaa acatttccta ttccaaaagc ttttgttggc
     3241 aatcagctac cttttgtagg atttacatat tatagcaatc gtagatactt accttcagca
     3301 aatgccagtg agaacagatc tagctccaat gtggataaaa gcttgcagga aagtttacag
     3361 aaaacaatct acaagcttga agagcaactg cataatgaaa tgcaattaaa agatgaaatg
     3421 gagcagaaat gcagaacttc aaacttaaaa ctagacaaga taatgaaaga attggatgaa
     3481 gaggggaatc aaagaagaaa tctagaatct gcagtgtctc aaattgagaa agaaaagatg
     3541 ttgctacaac atagaattaa tgagtaccaa agaaaagtgg aacaggaaaa tgagaagaga
     3601 agaaatatag aaaatgaagt ttctacacta aaggaccagt tagaagactt acggaaagca
     3661 agtcagacct cacagcttgc taatgagaag ctgacacagt tacagaagca gttagaagaa
     3721 gcgaatgact tacttaggac agaatcagac acagctgtaa gattaaggaa gagtcacaca
     3781 gagatgagca agtcaattag tcagctagag tccctgaaca gagaattaca ggaaagaaac
     3841 agaattttag agaattccaa gtcacaagca gacaaggatt actaccagct gcaagctgta
     3901 ttggaagctg agcgaagaga cagagggcat gattctgaga tgattggaga ccttcaagca
     3961 cgaattacat ctttacaaga agaagtgaaa catctcaaac ataatcttga aagagtggaa
     4021 ggagaaagaa aagaggctca agacatgctt aatcattcag aaaaggaaaa gaataatttg
     4081 gagatagatt tgaattacaa gctcaagtct atacaacagc ggttagaaca agaagtaaat
     4141 gaacataaag tcaccaaagc acgcctaact gacaagcacc aatctatcga agaggcaaag
     4201 tctgttgcca tgtgtgaaat ggaaaaaaag ctgaaggaag agagagaagc tcgagagaag
     4261 gcagaaaata gagttgttga gactgagaaa caatgttcca tgctagatgt tgacctgaaa
     4321 cagtctcagc agaagctaga acatttgacc gaaaataaag aaaggatgga ggatgaagtt
     4381 aagaatctcg ccctgcagct ggaacaggaa tcaaataaac ggctcttgtt acagaatgaa
     4441 ttgaagactc aagcatttga agcagacaat ttaaaaggtt tagaaaaaca gatgaaacag
     4501 gaaataaata ctttattgga agcaaagaga ttgttagaat ttgagttagc tcagcttaca
     4561 aagcaatata ggggaaatga agggcaaatg cgggagttac aagatcaact tgaagctgag
     4621 cagtatttct ctacacttta taaaacccaa gtaaaggagc ttaaagaaga aattgaagaa
     4681 aaaaaccgag aaaatttaag gaaaatacag gaactgcaaa gtgaaaaaga aactctctct
     4741 actcagttgg atttagcaga aacaaaagct gagtctgagc agctggcgcg agggattctg
     4801 gaagaacagt attttgagtt gactcaggaa agcaagaaag ctgcttcaag aaatagacaa
     4861 gagattacag ataaagatca tactgttagt cggcttgaag aaacaaacag tgtgctaacc
     4921 aaagatattg aaatgctaag aaaagaaaat gaagagctaa atgaaagaat gaggacagca
     4981 gaagaagaat ataaattgaa gaaggaggaa gaaataaata atcttaaggc tgcttttgaa
     5041 aagaatatca gtaccgaacg gacccttaaa acacaggctg ttaacaagtt ggcagaaata
     5101 atgaatcgca aagattttaa aattgatcga aagaaagcta atacacaaga tttaagaaaa
     5161 aaagaaaagg aaaatagaaa gctacaactg gaacttaacc aagagagaga aaaatttaac
     5221 caaatggtgg tgaaacacca gaaggagctg aatgacatgc aagcgcaatt ggtagaagaa
     5281 tgtacacata gaaatgaact tcaaatgcag ttggctagca aagagagtga tattgagcag
     5341 ttgcgggcaa aacttttgga cctttcggat tcaactagtg ttgctagttt tcctagtgca
     5401 gatgaaactg atggaaacct tccagagtca agaattgaag gttggctttc tgtaccaaat
     5461 agaggaaata tcaaaagata tggctggaag aaacagtatg ttgtggtaag cagcaaaaag
     5521 atcttgttct ataatgatga acaagataag gaacaatcca gtccatctat ggtgttagac
     5581 atagataaat tgtttcatgt ccgacctgta acccaaggag atgtgtacag agcagaaact
     5641 gaagaaattc ctaaaatatt ccagatacta tatgcaaatg aaggtgaatg tagaaaagat
     5701 atagaagtgg aaccggttca acaaggtgaa aaaactaatt tccagaatca caaaggccat
     5761 gaattcattc ctaccctcta ccactttcct gccaactgtg aggcctgtgc caaacctttg
     5821 tggcatgtct ttaagccacc acctgcccta gagtgtcgaa gatgccatgt taagtgccac
     5881 agagaccatc tagacaagaa agaggacttg atttccccgt gcaaagtaag ttacgatgta
     5941 acatcagcaa gagatatgct gctgttagca tgttctcagg atgaacaaaa aaagtgggtt
     6001 actcatctag taaagaagat tcctaagaac ccaccatctg gctttgtccg tgcctctcct
     6061 cgaacgctgt caacaagatc cactgcaaat cagtctttcc gcaaagtagt caaaaataca
     6121 tctggaaaaa ctagttaagg taccgcgggc ccgggatcca ccggatctag ataactgatc
     6181 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     6241 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     6301 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     6361 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag
     6421 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     6481 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     6541 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     6601 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     6661 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     6721 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     6781 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     6841 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     6901 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6961 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     7021 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     7081 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     7141 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     7201 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     7261 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     7321 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     7381 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     7441 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     7501 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     7561 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     7621 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     7681 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     7741 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     7801 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     7861 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     7921 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     7981 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     8041 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     8101 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     8161 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     8221 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     8281 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     8341 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     8401 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     8461 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     8521 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     8581 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     8641 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     8701 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     8761 tcagaccccg tagaa
//
