LOCUS       Exported                7874 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Mammalian expression of mRuby3-Galectin8 (Gal8) fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    PB-CAG-mRuby3-Gal8-P2A-Zeo
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7874)
  TITLE     A Genetically Encoded Sensor of Endosomal Disruption Demonstrates 
            Efficient Entry to the Cytosol Using Polymeric Nanoparticles for 
            mRNA Delivery In Vitro and In Vivo
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7874)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7874
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    complement(314..347)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     enhancer        443..746
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        748..1023
                     /label=chicken beta-actin promoter
     intron          1024..2040
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1964..1986
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2048..2067
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2133..2142
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2139..2849
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     CDS             3945..4001
                     /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"
     primer_bind     complement(4472..4491)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4537..4592
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4591..4610)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   5263..5297
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5320..5336)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5320..5336)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5333..5355)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5344..5360
                     /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(5368..5398)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5413..5434
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5493..5688
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5539..5674
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5601..5620
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    5694..5828
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5731..5750)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5785..5804
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5896..5913)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6067..6655)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6147..6166)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6826..7686)
                     /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     7449..7468
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7687..7791)
                     /gene="bla"
                     /label=AmpR promoter
     repeat_region   7829..17
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 cgtaaaattg acgcatgtgt tttatcgatc tgtatatcga ggtttattta ttaatttgaa
       61 tagatattaa gttttattat atttacactt acatactaat aataaattca acaaacaatt
      121 tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta
      181 acaaaacttt taaacattct ctcttttaca aaaataaact tattttgtac tttaaaaaca
      241 gtcatgttgt attataaaat aagtaattag cttaacttat acataataga aacaaattat
      301 acttattaat cgcataactt cgtataatgt atgctatacg aagttatgcg attaagggat
      361 ctgtagggcg cagtagtcca actagttatt aatagtaatc aattacgggg tcattagttc
      421 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      541 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      601 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      661 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      721 acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc
      781 ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg
      841 cagcgatggg ggcggggggg gggggggggc gcgccaggcg gggcggggcg gggcgagggg
      901 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
      961 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
     1021 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
     1081 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
     1141 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
     1201 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
     1261 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
     1321 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     1381 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     1441 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     1501 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     1561 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     1621 gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc
     1681 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     1741 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     1801 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     1861 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     1921 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1981 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     2041 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attctgcagt
     2101 cgacggtacc gcgggcccgg gatccaccgg tcgccaccat ggtgtctaag ggcgaagagc
     2161 tgatcaagga aaatatgcgt atgaaggtgg tcatggaagg ttcggtcaac ggccaccaat
     2221 tcaaatgcac aggtgaagga gaaggcagac cgtacgaggg agtgcaaacc atgaggatca
     2281 aagtcatcga gggaggaccc ctgccatttg cctttgacat tcttgccacg tcgttcatgt
     2341 atggcagccg tacctttatc aagtacccgg ccgacatccc tgatttcttt aaacagtcct
     2401 ttcctgaggg ttttacttgg gaaagagtta cgagatacga agatggtgga gtcgtcaccg
     2461 tcacgcagga caccagcctt gaggatggcg agctcgtcta caacgtcaag gtcagagggg
     2521 taaactttcc ctccaatggt cccgtgatgc agaagaagac caagggttgg gagcctaata
     2581 cagagatgat gtatccagca gatggtggtc tgagaggata cactgacatc gcactgaaag
     2641 ttgatggtgg tggccatctg cactgcaact tcgtgacaac ttacaggtca aaaaagaccg
     2701 tcgggaacat caagatgccc ggtgtccatg ccgttgatca ccgcctggaa aggatcgagg
     2761 agagtgacaa tgaaacctac gtagtgcaaa gagaagtggc agttgccaaa tacagcaacc
     2821 ttggtggtgg catggacgag ctgtacaagg acatgtccat gatgttgtcc ttaaacaacc
     2881 tacagaatat catctataac ccggtaatcc cgtttgttgg caccattcct gatcagctgg
     2941 atcctggaac tttgattgtg atacgtgggc atgttcctag tgacgcagac agattccagg
     3001 tggatctgca gaatggcagc agcatgaaac ctcgagccga tgtggccttt catttcaatc
     3061 ctcgtttcaa aagggccggc tgcattgttt gcaatacttt gataaatgaa aaatggggac
     3121 gggaagagat cacctatgac acgcctttca aaagagaaaa gtcttttgag atcgtgatta
     3181 tggtgctgaa ggacaaattc caggtggctg taaatggaaa acatactctg ctctatggcc
     3241 acaggatcgg cccagagaaa atagacactc tgggcattta tggcaaagtg aatattcact
     3301 caattggttt tagcttcagc tcggacttac aaagtaccca agcatctagt ctggaactga
     3361 cagagataag tagagaaaat gttccaaagt ctggcacgcc ccagcttcct agtaatagag
     3421 gaggagacat ttctaaaatc gcacccagaa ctgtctacac caagagcaaa gattcgactg
     3481 tcaatcacac tttgacttgc accaaaatac cacctatgaa ctatgtgtca aagaggctgc
     3541 cattcgctgc aaggttgaac acccccatgg gccctggacg aactgtcgtc gttaaaggag
     3601 aagtgaatgc aaatgccaaa agctttaatg ttgacctact agcaggaaaa tcaaaggata
     3661 ttgctctaca cttgaaccca cgcctgaata ttaaagcatt tgtaagaaat tcttttcttc
     3721 aggagtcctg gggagaagaa gagagaaata ttacctcttt cccatttagt cctgggatgt
     3781 actttgagat gataatttat tgtgatgtta gagaattcaa ggttgcagta aatggcgtac
     3841 acagcctgga gtacaaacac agatttaaag agctcagcag tattgacacg ctggaaatta
     3901 atggagacat ccacttactg gaagtaagga gctggggaag cggagctact aacttcagcc
     3961 tgctgaagca ggctggagac gtggaggaga accctggacc tataatggcc aagttgacca
     4021 gtgctgtccc agtgctcaca gccagggatg tggctggagc tgttgagttc tggactgaca
     4081 ggttggggtt ctccagagat tttgtggagg atgactttgc aggtgtggtc agagatgatg
     4141 tcaccctgtt catctcagca gtccaggacc aggtggtgcc tgacaacacc ctggcttggg
     4201 tgtgggtgag aggactggat gagctgtatg ctgagtggag tgaggtggtc tccaccaact
     4261 tcagggatgc cagtggccct gccatgacag agattggaga gcagccctgg gggagagagt
     4321 ttgccctgag agacccagca ggcaactgtg tgcactttgt ggcagaggag caggactgat
     4381 tctagagcgg ccgcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg
     4441 ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg
     4501 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     4561 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     4621 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg
     4681 ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg
     4741 tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt
     4801 ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat
     4861 ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc
     4921 tcgacctgca gagctccagc ctacacaatc gctcaagacg tgtaatgctt ttattatata
     4981 ttagtcacga tatctataac aagaaaatat atatataata agttatcacg taagtagaac
     5041 atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc
     5101 atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg
     5161 acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg
     5221 gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc
     5281 agactatctt tctagggtta aaagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     5341 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5401 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5461 ccagtcggga aacctgtcgt gccagcggat ccgcatctca ataagtcagc aaccatagtc
     5521 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     5581 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     5641 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctaacttgt
     5701 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5761 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5821 tctggatccg ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5881 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5941 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6001 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6061 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6121 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6181 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6241 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6301 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6361 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6421 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6481 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     6541 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6601 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6661 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6721 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6781 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6841 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6901 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6961 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7021 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7081 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7141 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7201 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7261 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7321 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7381 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7441 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7501 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7561 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7621 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7681 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     7741 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7801 ccgaaaagtg ccacctgggt cgacttaacc ctagaaagat aatcatattg tgacgtacgt
     7861 taaagataat catg
//
