LOCUS       pRK5_V5-nb-KS           6026 bp    DNA     circular SYN 13-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6026)
  TITLE     Spatial spread of Ras activity
REFERENCE   2  (bases 1 to 6026)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 13, 2025 from SnapGene 8.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pRK5_V5-nb-KS
FEATURES             Location/Qualifiers
     source          1..6026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          2253..2303
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     902..971
                     /label=HN181
     primer_bind     907..941
                     /label=HN411
     regulatory      933..941
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             939..1646
                     /codon_start=1
                     /label=mCherry (full)
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1258..1278)
                     /label=backbone_R
     primer_bind     complement(1622..1673)
                     /label=HN423
     misc_feature    1622..1646
                     /label=Feature 26
     misc_feature    1647..1673
                     /label=Feature 28
     misc_feature    1656..1775
                     /label=Feature 31
     primer_bind     1656..1775
                     /label=HN425
     CDS             1656..1712
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1722..1775
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(1756..1797)
                     /label=HN440
     misc_feature    1756..1775
                     /label=Feature 32
     primer_bind     1758..1827
                     /label=HN424
     misc_feature    1758..1802
                     /label=Feature 30
     primer_bind     1758..1801
                     /label=HN441
     CDS             1776..1802
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    1803..1827
                     /label=Feature 29
     CDS             1803..1823
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1824..1853
                     /codon_start=1
                     /label=GGGGSx2-linker1
                     /translation="GGGGSGGGGS"
     primer_bind     1832..1875
                     /label=HN863
     primer_bind     complement(1835..1875)
                     /label=HN864
     misc_feature    1835..1853
                     /label=Feature 33
     CDS             1854..2222
                     /codon_start=1
                     /label=V5-nanobody
                     /translation="MAEVQLQASGGGFVQPGGSLRLSCAASGTTSFGDTMGWFRQAPGK
                     EREFVSAISRQGDKHYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAEWMNT
                     RREFITPYWGQGTQVTVSS"
     CDS             2223..2252
                     /codon_start=1
                     /label=GGGGSx2-linker2
                     /translation="GGGGSGGGGS"
     CDS             2253..2303
                     /codon_start=1
                     /label=KS
                     /translation="MMMMNKLVLAQFFFSCL"
     primer_bind     complement(2280..2328)
                     /label=HN280
     regulatory      2326..2335
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2339..2473
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2376..2395)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2430..2449
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2542..2899
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(2565..2585)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2750..2885
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2812..2831
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2835..2846)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2919..2936)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2919..2935)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2928..2950)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3148..3603
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3235..3254)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3445..3466
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3620..3639)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3739..3761
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3799..3817)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3885..3989
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3990..4058,4059..4850)
                     /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(4208..4227)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5021..5609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5510..5529
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5763..5780
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(5933..5938,5939..5956,5957..5963)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5971..5987
                     /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     5976..5998
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5995..6011
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5995..6011
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcgtaccg gtgccaccat ggtttccaaa ggagaggagg
      961 ataatatggc tataattaaa gagtttatgc ggttcaaggt gcatatggag ggatctgtga
     1021 acggtcacga gttcgaaatc gagggcgaag gcgaggggcg accctatgaa gggacacaga
     1081 cggccaaact gaaggtgacc aagggtggcc ccctgccttt cgcctgggac atcctgtcac
     1141 cccagtttat gtacggctct aaggcttacg ttaagcaccc tgccgatatt ccggactact
     1201 tgaaactgag cttccctgaa gggttcaagt gggaacgggt aatgaatttt gaggatggcg
     1261 gtgtagtcac agttacccag gacagctctc ttcaggacgg agaatttatc tataaggtta
     1321 aacttcgggg cactaacttc ccatcagacg gccccgtgat gcagaagaaa actatgggct
     1381 gggaagccag ctcagaacgc atgtatcccg aggacggagc actgaaggga gaaatcaagc
     1441 agcgactcaa gctgaaggat gggggacatt atgatgctga ggtcaaaacc acctataagg
     1501 ccaagaaacc cgttcagctt cctggggcat acaacgtgaa tatcaaactg gatattacat
     1561 cccataacga agactatacc atcgtggaac agtacgagcg ggccgagggc agacatagca
     1621 cagggggcat ggatgaattg tacaagggaa gcggagctac taacttcagc ctgctgaagc
     1681 aggctggaga cgtggaggag aaccctggac ctggaagcgg agagggcaga ggaagtcttc
     1741 taacatgcgg tgacgtggag gagaatcccg gcccatatcc gtacgacgta ccagactacg
     1801 cacctaagaa gaagaggaag gttgggggag gcggcagtgg cggaggggga tctatggctg
     1861 aagtgcagct gcaagccagc ggcggaggct tcgtgcagcc cggcggatct ctgagactgt
     1921 cttgtgccgc ttctggcacc accagcttcg gcgatacaat gggctggttc cggcaggccc
     1981 ctggaaagga aagagagttt gtgtccgcca tctccagaca gggcgacaag cactactatg
     2041 ccgacagcgt gaaaggcaga ttcacaatca gccgggacaa cagcaagaac accgtgtacc
     2101 tgcagatgaa ctctctgaga gccgaagata ccgccaccta ctactgcgcc gagtggatga
     2161 ataccagacg cgagttcatc accccttact ggggccaggg aacacaggtg accgtgtcta
     2221 gcggcggcgg aggaagcgga ggcggaggat ctatgatgat gatgaataag ttggttctag
     2281 cgcagttctt cttctcctgt ctataggtcg acctgcagaa gcttggccgc catggcccaa
     2341 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2401 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     2461 tcatgtctgg atcgggaatt aattcggcgc agcaccatgg cctgaaataa cctctgaaag
     2521 aggaacttgg ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     2581 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     2641 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2701 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     2761 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     2821 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     2881 aggcctaggc ttttgcaaaa agctgttaac agcttggcac tggccgtcgt tttacaacgt
     2941 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     3001 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     3061 ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3121 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     3181 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3241 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3301 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3361 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3421 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3481 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     3541 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     3601 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     3661 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3721 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3781 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     3841 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3901 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3961 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     4021 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     4081 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     4141 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     4201 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     4261 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     4321 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     4381 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     4441 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     4501 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     4561 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     4621 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     4681 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     4741 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4801 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4861 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4921 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4981 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5041 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5101 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5161 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5221 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5281 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5341 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5401 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5461 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     5521 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     5581 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5641 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5701 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5761 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     5821 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     5881 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     5941 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     6001 acagctatga catgattacg aattaa
//
