LOCUS       sequence-139200-        5301 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 5301)
  TITLE     Bokoch Lab GTPases
REFERENCE   2  (bases 1 to 5301)
  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"
COMMENT     Sequence Label: pRK5-myc-RhoA-Q63L
FEATURES             Location/Qualifiers
     source          1..5301
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    55..76
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(91..96,97..114,115..121)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    129..145
                     /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     134..156
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     153..169
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     153..169
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        198..577
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        578..781
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     732..752
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     778..802
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1012..1030
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1012..1029
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1043..1062
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1109..1118
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1118..1147
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             join(1184..1369,1370..1372,1373..1765)
                     /codon_start=1
                     /product="constitutively active mutant of human Rho family 
                     small GTPase RhoA"
                     /label=RhoA (Q63L)
                     /translation="MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGLEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     regulatory      1785..1794
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1798..1932
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1835..1854)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1889..1908
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2001..2358
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2001..2192
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2024..2044)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2209..2344
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2378..2395)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2378..2394)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2387..2409)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2607..3062
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2694..2713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2904..2925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3079..3098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3198..3220
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3258..3276)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3344..3448
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3449..3517,3518..4309)
                     /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(3667..3686)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4480..5068
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4969..4988
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5222..5239
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
       61 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      121 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgaca tgattacgaa
      181 ttaattcgag ctcgcccgac attgattatt gactagttat taatagtaat caattacggg
      241 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      301 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      361 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      421 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      481 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      541 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      601 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      661 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      721 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      781 tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag
      841 aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg tgcattggaa cgcggattcc
      901 ccgtgccaag agtgacgtaa gtaccgccta tagagtctat aggcccaccc ccttggcttc
      961 gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg
     1021 acactataga ataacatcca ctttgccttt ctctccacag gtgtccactc ccaggtccaa
     1081 ctgcacctcg gttctatcga ttgaattggc caccatggag cagaagctga tctccgagga
     1141 ggacctggga tccatggagt atgatgttcc tgattatgct catatggctg ccatccggaa
     1201 gaaactggtg attgttggtg atggagcctg tggaaagaca tgcttgctca tagtcttcag
     1261 caaggaccag ttcccagagg tgtatgtgcc cacagtgttt gagaactatg tggcagatat
     1321 cgaggtggat ggaaagcagg tagagttggc tttgtgggac acagctgggc tggaagatta
     1381 tgatcgcctg aggcccctct cctacccaga taccgatgtt atactgatgt gtttttccat
     1441 cgacagccct gatagtttag aaaacatccc agaaaagtgg accccagaag tcaagcattt
     1501 ctgtcccaac gtgcccatca tcctggttgg gaataagaag gatcttcgga atgatgagca
     1561 cacaaggcgg gagctagcca agatgaagca ggagccggtg aaacctgaag aaggcagaga
     1621 tatggcaaac aggattggcg cttttgggta catggagtgt tcagcaaaga ccaaagatgg
     1681 agtgagagag gtttttgaaa tggctacgag agctgctctg caagctagac gtgggaagaa
     1741 aaaatctggt tgccttgtct tgtgagaatt cctgcagaag cttggccgcc atggcccaac
     1801 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     1861 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     1921 catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac ctctgaaaga
     1981 ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
     2041 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2101 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2161 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     2221 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     2281 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     2341 ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc
     2401 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     2461 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     2521 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2581 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     2641 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2701 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2761 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2821 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2881 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2941 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3001 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     3061 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     3121 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3181 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3241 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     3301 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3361 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3421 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3481 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3541 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3601 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3661 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3721 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3781 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3841 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3901 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3961 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     4021 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     4081 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4141 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     4201 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4261 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4321 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4381 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4441 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4501 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4561 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4621 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4681 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4741 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4801 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4861 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4921 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4981 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5041 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5101 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5161 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5221 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     5281 gcgttggccg attcattaat g
//
