LOCUS       pRK5_mCherry-P2A        5444 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 5444)
  TITLE     Spatial spread of Ras activity
REFERENCE   2  (bases 1 to 5444)
  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_mCherry-P2A.dna
FEATURES             Location/Qualifiers
     source          1..5444
                     /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
     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
     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"
     regulatory      1744..1753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1757..1891
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1794..1813)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1848..1867
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1960..2317
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(1983..2003)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2168..2303
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2230..2249
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2253..2264)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2337..2354)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2337..2353)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2346..2368)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2566..3021
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2653..2672)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2863..2884
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3038..3057)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3157..3179
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3217..3235)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3303..3407
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3408..3476,3477..4268)
                     /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(3626..3645)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4439..5027
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4928..4947
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5181..5198
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(5351..5356,5357..5374,5375..5381)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5389..5405
                     /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     5394..5416
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5413..5429
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5413..5429
                     /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 ataggtcgac ctgcagaagc
     1741 ttggccgcca tggcccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1801 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1861 actcatcaat gtatcttatc atgtctggat cgggaattaa ttcggcgcag caccatggcc
     1921 tgaaataacc tctgaaagag gaacttggtt aggtaccttc tgaggcggaa agaaccagct
     1981 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2041 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     2101 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     2161 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     2221 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     2281 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctgttaacag cttggcactg
     2341 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     2401 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     2461 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
     2521 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     2581 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2641 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2701 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2761 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     2821 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2881 ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga
     2941 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3001 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     3061 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3121 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3181 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     3241 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     3301 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3361 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3421 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3481 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     3541 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     3601 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     3661 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     3721 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     3781 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     3841 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     3901 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     3961 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4021 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4081 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     4141 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     4201 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     4261 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     4321 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4381 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4441 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4501 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4561 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4621 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4681 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4741 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4801 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4861 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4921 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4981 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5041 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5101 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     5161 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     5221 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     5281 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     5341 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     5401 aacaatttca cacaggaaac agctatgaca tgattacgaa ttaa
//
