LOCUS       sequence-471591-        6680 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 6680)
  AUTHORS   Subauste MC, Von Herrath M, Benard V, Chamberlain CE, Chuang TH, Chu
            K, Bokoch GM, Hahn KM
  TITLE     Rho family proteins modulate rapid apoptosis induced by cytotoxic T 
            lymphocytes and Fas.
  JOURNAL   J Biol Chem. 2000 Mar 31. 275(13):9725-33.
  PUBMED    10734125
REFERENCE   2  (bases 1 to 6680)
  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: "J Biol 
            Chem. 2000 Mar 31. 275(13):9725-33."
COMMENT     Sequence Label: pcDNA3-EGFP-RhoA-Q63L
FEATURES             Location/Qualifiers
     source          1..6680
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     377..396
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(615..719)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(794..813)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        985..1364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1365..1568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1519..1539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1613..1632
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1613..1631
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1645..1654
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1651..1653,1654..1656,1657..2367)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1651..1653,1654..1656,1657..2367)
                     /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(1696..1717)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1957..1976)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2304..2325
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(2374..2559,2560..2562,2563..2955)
                     /codon_start=1
                     /product="constitutively active mutant of human Rho family 
                     small GTPase RhoA"
                     /label=RhoA (Q63L)
                     /translation="MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGLEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     promoter        complement(2983..3001)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2984..3001)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3021..3038)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3027..3251
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3297..3725
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3384..3403)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3594..3615
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3734..3754)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3739..4068
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3919..4054
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3981..4000
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4135..4929
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4189..4208)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4799..4818
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5103..5224
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5140..5159)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5194..5213
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5273..5289)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5273..5289)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5286..5308)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5297..5313
                     /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)."
     promoter        complement(join(5321..5327,5328..5345,5346..5351))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5366..5387
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5504..5521)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5675..6263)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5755..5774)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6434..6680,1..545,546..614))
                     /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"
ORIGIN
        1 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
       61 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      121 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      181 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
      241 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
      301 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      361 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      421 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
      481 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
      541 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
      601 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
      661 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
      721 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
      781 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
      841 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
      901 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
      961 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
     1021 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1081 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1141 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1201 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1261 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1321 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     1381 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     1441 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     1501 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     1561 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
     1621 ctcactatag ggagacccaa gcttgccacc atggtgagca agggcgagga gctgttcacc
     1681 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1741 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1801 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1861 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1921 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1981 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2041 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     2101 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2161 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2221 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     2281 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2341 actctcggca tggacgagct gtacaaggaa ttcatggctg ccatccggaa gaaactggtg
     2401 attgttggtg atggagcctg tggaaagaca tgcttgctca tagtcttcag caaggaccag
     2461 ttcccagagg tgtatgtgcc cacagtgttt gagaactatg tggcagatat cgaggtggat
     2521 ggaaagcagg tagagttggc tttgtgggac acagctgggc tggaagatta tgatcgcctg
     2581 aggcccctct cctacccaga taccgatgtt atactgatgt gtttttccat cgacagccct
     2641 gatagtttag aaaacatccc agaaaagtgg accccagaag tcaagcattt ctgtcccaac
     2701 gtgcccatca tcctggttgg gaataagaag gatcttcgga atgatgagca cacaaggcgg
     2761 gagctagcca agatgaagca ggagccggtg aaacctgaag aaggcagaga tatggcaaac
     2821 aggattggcg cttttgggta catggagtgt tcagcaaaga ccaaagatgg agtgagagag
     2881 gtttttgaaa tggctacgag agctgctctg caagctagac gtgggaagaa aaaatctggt
     2941 tgccttgtct tgtgactcga gcatgcatct agagggccct attctatagt gtcacctaaa
     3001 tgctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3061 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3121 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3181 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3241 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3301 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3361 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3421 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3481 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3541 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3601 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     3661 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3721 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     3781 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     3841 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     3901 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     3961 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     4021 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     4081 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4141 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4201 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4261 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4321 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4381 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4441 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4501 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4561 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     4621 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     4681 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     4741 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     4801 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4861 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4921 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4981 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5041 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5101 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5161 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5221 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5281 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5341 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5401 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5461 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5521 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5581 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5641 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5701 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5761 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5821 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5881 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5941 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6001 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6061 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6121 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6181 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6241 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6301 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6361 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6421 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6481 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6541 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6601 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6661 ccgcctccat ccagtctatt
//
