LOCUS       sequence-159422-        7323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7323)
  AUTHORS   Pertz O, Hodgson L, Klemke RL, Hahn KM
  TITLE     Spatiotemporal dynamics of RhoA activity in migrating cells.
  JOURNAL   Nature. 2006 Apr 20. 440(7087):1069-72.
  PUBMED    16547516
REFERENCE   2  (bases 1 to 7323)
  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"; journalName: "Nature. 
            2006 Apr 20. 440(7087):1069-72."
COMMENT     Sequence Label: pTriEx-RhoA FLARE.sc Biosensor Q63L
FEATURES             Location/Qualifiers
     source          1..7323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    11..44
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_recomb     53..758
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     CDS             53..641
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     3'UTR           298..670
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     terminator      complement(771..818)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     888..907
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    906..961
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     1007..1026
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(1116..1139)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             complement(join(1149..1541,1542..1544,1545..1730))
                     /codon_start=1
                     /product="constitutively active mutant of human Rho family 
                     small GTPase RhoA"
                     /label=RhoA (Q63L)
                     /translation="MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGLEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     CDS             complement(join(1737..2447,2448..2450,2451..2453))
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2526..3236,3237..3239,3240..3242))
                     /codon_start=1
                     /product="cyan variant of GFP"
                     /label=CFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(3507..3524)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        complement(3545..3654)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     protein_bind    3670..3694
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(3694..3713)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3695..3713)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3755..3774)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(3836..3858)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(4061..4263)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4090..4110)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4265..4568
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_recomb     4636..5463
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             4696..5301
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     CDS             complement(5339..5463)
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             join(5706..5774,5775..6566)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5924..5943)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6579..7166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7068..7087
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttccgagctt ataacttcgt ataatgtatg ctatacgaag ttatgcgatc gcaattccaa
       61 aaagctcaac tacaaatttg atcgcggacg tgttagccga cacaattaat aggcgtcgtg
      121 tggctatggc aaaatcgtct tcggaagcaa cttctaacga cgagggttgg gacgacgacg
      181 ataatcggcc taataaagct aacacgcccg atgttaaata tgtccaagct ctatttaacg
      241 tgtttacgtc gagtcaattg tacactaacg acagtgatga aagaaataca aaagcgcata
      301 atattttgaa cgacgtcgaa cctttattac aaaacaaaac acaaacgaat atcgacaaag
      361 ctagattgct gctacaagat ttggcaagtt ttgtggcgtt gagcgaaaat ccattagata
      421 gtccagccat cggttcggaa aaacaaccct tgtttgaaac taatcgaaac ctattttaca
      481 aatctattga ggatttaata tttaaattca gatataaaga cgctgaaaat catttgattt
      541 tcgctctaac ataccaccct aaagattata aatttaatga attattaaaa tacgtacaac
      601 aattgtctgt aaatcaacaa cgcacagaat ctagcgctta ataaatgtac taataacaat
      661 gtatcgtgtt ttaatacgcc ggaccagtga acagaggtgc gtctggtgca aactccttta
      721 ctttgaacac cagggaaact tcaaggagaa tttcctccgc atgctttcag caaaaaaccc
      781 ctcaagaccc gtttagaggc cccaaggggt tatgctagtt acatatgggc atatgttgcc
      841 aaactctaaa ccaaatactc attctgatgt tttaaatgat ttgccctccc atatgtcctt
      901 ccgagtgaga gacacaaaaa attccaacac actattgcaa tgaaaataaa tttcctttat
      961 tagccagaag tcagatgctc aaggggcttc atgatgtccc cataattttt ggcagaggga
     1021 aaaagatcga tctcagtggt atttgtgagc cagggcattg gccacaccag ccaccacctt
     1081 ctgataggca gcctgcacct gaggttaatc acttagtgat ggtgatggtg atggtggtgc
     1141 tcgagttatc acaagacaag gcaaccagat tttttcttcc cacgtctagc ttgcagagca
     1201 gctctcgtag ccatttcaaa aacctctctc actccatctt tggtctttgc tgaacactcc
     1261 atgtacccaa aagcgccaat cctgtttgcc atatctctgc cttcttcagg tttcaccggc
     1321 tcctgcttca tcttggctag ctcccgcctt gtgtgctcat cattccgaag atccttctta
     1381 ttcccaacca ggatgatggg cacgttggga cagaaatgct tgacttctgg ggtccacttt
     1441 tctgggatgt tttctaaact atcagggctg tcgatggaaa aacacatcag tataacatcg
     1501 gtatctgggt aggagagggg cctcaggcga tcataatctt ccagcccagc tgtgtcccac
     1561 aaagccaact ctacctgctt tccatccacc tcgatatctg ccacatagtt ctcaaacact
     1621 gtgggcacat acacctctgg gaactggtcc ttgctgaaga ctatgagcaa gcatgtcttt
     1681 ccacaggctc catcaccaac aatcaccagt ttcttccgga tggcagccat tccccccttg
     1741 tacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag caggaccatg
     1801 tgatcgcgct tctcgttggg gtctttgctc agggcggact ggtagctcag gtagtggttg
     1861 tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc
     1921 tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat gccgttcttc
     1981 tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc
     2041 aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg
     2101 tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg
     2161 gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg
     2221 tggtcggggt agcgggcgaa gcacatcagg ccgtagccga aggtggtcac gagggtgggc
     2281 cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg
     2341 gcatcgccct cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc gccgtccagc
     2401 tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac catagcggcc
     2461 gcgccactac cacctttagt agaaccttca ccagaaccag gtttaccaga accagaagta
     2521 gatcccttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     2581 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg gtagctcagg
     2641 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     2701 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtg ggccttgatg
     2761 ccgttcttct gcttgtcggc ggtgatatag acgttgtggc tgatgtagtt gtactccagc
     2821 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
     2881 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
     2941 aagaagatgg tacgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
     3001 tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cccaggtcag ggtggtcacg
     3061 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
     3121 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acctgtggcc gtttacgtcg
     3181 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
     3241 attcccccgc ctgtcttctc cagcacctgg gcctcctttc gccgctgcag ctcacccatg
     3301 tagctgagaa tacggctgtt gcacaccagc aggctcttgg tggcttccag agcctgctct
     3361 cgctgggagc aggctgccag cagcttgcag gccccatccc tcatccggat ctcatgatct
     3421 agtttcctct gcagctctgt gtcctccagg ctgagtgcca tctgccggat gtagagcata
     3481 ttgaggtcct ccaggatgcc actaccgtga tggtggtgat ggtgtgccat ggtatatctc
     3541 ctttgattgt aaataaaatg taatttacag tatagtattt taattaatat acaaatgatt
     3601 tgataataat tcttatttaa ctataatata ttgtgttggg ttgaattaaa ggtcccggat
     3661 taactccggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgg
     3721 tccgatccaa ttctttgcca aaatgatgag acagcacaat aaccagcacg ttgcccagga
     3781 gctgtaggaa aaagaagaag gcatgaacat ggttagcaga ggctctagag ccgccggtca
     3841 cacgccagaa gccgaacccc gccctgcccc gtcccccccg aaggcagccg tccccccgcg
     3901 gacagccccg agccgctccc cccgcacaaa gggcccgata cttacctgcc cagtgcctca
     3961 cgaccaactt ctgcacgtta agttcgagac tggcgttagc aatttaactg tgataaacta
     4021 ccgcaataaa gcatctagtg atctgacggt tcactaaacg acgtctgctt atatagacct
     4081 cccaccgtac acgcctaccg cccatttgcg tcaacggggc ggggttatta cgacattttg
     4141 gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac
     4201 ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt ggtgtactgc caaaaccgca
     4261 tcagcatggt aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccgta
     4321 aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg
     4381 gcggacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact
     4441 ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta
     4501 ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt
     4561 atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt
     4621 aataggccgg ccatcagcaa ctatatattg atagacattt ccagtttgtg atattagttt
     4681 gtgcgtctca ttacaatggc tgttattttt aacaacaaac aactgctcgc agacaatagt
     4741 atagaaaagg gaggtgaact gtttttgttt aacggttcgt acaacatttt ggaaagttat
     4801 gttaatccgg tgctgctaaa aaatggtgta attgaactag aagaagctgc gtactatgcc
     4861 ggcaacatat tgtacaaaac cgacgatccc aaattcattg attatataaa tttaataatt
     4921 aaagcaacac actccgaaga actaccagaa aatagcactg ttgtaaatta cagaaaaact
     4981 atgcgcagcg gtactataca ccccattaaa aaagacatat atatttatga caacaaaaaa
     5041 tttactctat acgatagata catatatgga tacgataata actatgttaa tttttatgaa
     5101 gagaaaaatg aaaaagagaa ggaatacgaa gaagaagacg acaaggcgtc tagtttatgt
     5161 gaaaataaaa ttatattgtc gcaaattaac tgtgaatcat ttgaaaatga ttttaaatat
     5221 tacctcagcg attataacta cgcgttttca attatagata atactacaaa tgttcttgtt
     5281 gcgtttggtt tgtatcgtta ataaaaaaca aatttgacat ttataattgt tttattattc
     5341 aataattaca aataggattg agacccttgc agttgccagc aaacggacag agcttgtcga
     5401 cagttttcat tttttggcag tttggtggtt tatacacatc ttgggatttg attaaaagat
     5461 gcatgacttc acccacacac ttggagtcgt ggttgtagaa atgcgtcaga gccttgatta
     5521 aacatcggtt ttcacacttg gcacacgaaa ccacgtttgc aatgtaacag tttagtataa
     5581 acctcttcct gtacataccg gcgcgtttat acacatcttg ggatttgatt aaaagatgca
     5641 ggaaacgcgc ggacatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5701 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5761 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5821 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5881 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5941 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     6001 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6061 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6121 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6181 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6241 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6301 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6361 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6421 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6481 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6541 gtgcctcact gattaagcat tggtaacaaa ggatcttctt gagatccttt ttttctgcgc
     6601 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6661 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6721 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6781 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6841 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6901 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6961 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     7021 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7081 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7141 tcgtcagggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7201 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7261 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     7321 gcg
//
