LOCUS       pRK5_TagBFP-P2A         5435 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 5435)
  TITLE     Spatial spread of Ras activity
REFERENCE   2  (bases 1 to 5435)
  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_TagBFP-P2A
FEATURES             Location/Qualifiers
     source          1..5435
                     /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..969
                     /label=HN525
     primer_bind     902..941
                     /label=HN181
     regulatory      933..938
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             939..1637
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(1611..1664)
                     /label=HN526
     CDS             1647..1703
                     /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      1735..1744
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1748..1882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1785..1804)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1839..1858
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1951..2308
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(1974..1994)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2159..2294
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2221..2240
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2244..2255)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2328..2345)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2328..2344)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2337..2359)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2557..3012
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2644..2663)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2854..2875
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3029..3048)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3148..3170
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3208..3226)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3294..3398
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3399..3467,3468..4259)
                     /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(3617..3636)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4430..5018
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4919..4938
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5172..5189
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(5342..5347,5348..5365,5366..5372)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5380..5396
                     /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     5385..5407
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5404..5420
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5404..5420
                     /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 gagcgagctg attaaggaga
      961 acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat
     1021 ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg
     1081 gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga
     1141 ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc cctgagggct
     1201 tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca
     1261 ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcacat
     1321 ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc gagacgctgt
     1381 accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc gtgggcggga
     1441 gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct aagaacctca
     1501 agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag gccaacaacg
     1561 agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc cctagcaaac
     1621 tggggcacaa gcttaatgga agcggagcta ctaacttcag cctgctgaag caggctggag
     1681 acgtggagga gaaccctgga cctggaagcg gataggtcga cctgcagaag cttggccgcc
     1741 atggcccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     1801 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     1861 tgtatcttat catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac
     1921 ctctgaaaga ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt
     1981 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2041 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     2101 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     2161 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     2221 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     2281 cttttttgga ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt
     2341 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     2401 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     2461 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
     2521 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     2581 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     2641 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     2701 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     2761 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     2821 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     2881 cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct
     2941 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     3001 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3061 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3121 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3181 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     3241 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     3301 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3361 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3421 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3481 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3541 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3601 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     3661 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3721 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     3781 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     3841 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     3901 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     3961 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4021 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4081 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4141 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4201 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4261 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4321 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4381 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4441 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4501 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4561 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4621 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4681 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4741 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4801 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4861 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4921 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4981 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5041 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5101 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5161 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     5221 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5281 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     5341 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     5401 acacaggaaa cagctatgac atgattacga attaa
//
