LOCUS       pRK5_V5-nb-noKS         5975 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 5975)
  TITLE     Spatial spread of Ras activity
REFERENCE   2  (bases 1 to 5975)
  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-noKS
FEATURES             Location/Qualifiers
     source          1..5975
                     /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
                     /label=V5-nanobody
     misc_feature    1854..1875
                     /label=Feature 32
     CDS             2223..2252
                     /codon_start=1
                     /label=GGGGSx2-linker2
                     /translation="GGGGSGGGGS"
     primer_bind     complement(2233..2277)
                     /label=HN284
     regulatory      2275..2284
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2288..2422
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2325..2344)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2379..2398
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2491..2848
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(2514..2534)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2699..2834
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2761..2780
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2784..2795)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2868..2885)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2868..2884)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2877..2899)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3097..3552
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3184..3203)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3394..3415
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3569..3588)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3688..3710
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3748..3766)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3834..3938
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3939..4007,4008..4799)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4157..4176)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4970..5558
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5459..5478
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5712..5729
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(5882..5887,5888..5905,5906..5912)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5920..5936
                     /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     5925..5947
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5944..5960
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5944..5960
                     /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 cttaggtcga cctgcagaag cttggccgcc
     2281 atggcccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2341 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2401 tgtatcttat catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac
     2461 ctctgaaaga ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt
     2521 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2581 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     2641 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     2701 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     2761 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     2821 cttttttgga ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt
     2881 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     2941 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     3001 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3061 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3121 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3181 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3241 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3301 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3361 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3421 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     3481 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3541 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3601 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3661 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3721 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3781 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3841 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3901 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3961 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4021 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4081 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4141 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4201 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4261 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4321 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4381 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4441 atgaagccat accaaacgac gagcgtgaca ccacgatgcc agcagcaatg gcaacaacgt
     4501 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4561 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4621 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4681 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4741 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4801 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4861 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4921 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4981 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5041 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5101 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     5161 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5221 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5281 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5341 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5401 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5461 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5521 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5581 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5641 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5701 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     5761 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5821 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     5881 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     5941 acacaggaaa cagctatgac atgattacga attaa
//
