LOCUS       Exported                8818 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Quadcistronic construct: Co-express four genes by three 2A peptides 
            -- T2A, P2A and E2A.
ACCESSION   .
VERSION     .
KEYWORDS    pGEMT-TPE2A-Mef2c-Tdtomato-Gata4-Tbx5
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8818)
  AUTHORS   Liu Z, Chen O, Wall JBJ, Zheng M, Zhou Y, Wang L, Ruth Vaseghi H, 
            Qian L, Liu J
  TITLE     Systematic comparison of 2A peptides for cloning multi-genes in a 
            polycistronic vector.
  JOURNAL   Sci Rep. 2017 May 19;7(1):2193. doi: 10.1038/s41598-017-02460-2.
  PUBMED    28526819
REFERENCE   2  (bases 1 to 8818)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8818
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1413..1466
                     /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"
     CDS             1473..2900
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     CDS             2916..2972
                     /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             4317..4376
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     promoter        complement(5983..6001)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5984..6001)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(6019..6035)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6019..6035)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6032..6054)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6043..6059
                     /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(6067..6097)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6112..6133
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6250..6267)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6421..7009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6501..6520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7180..8040)
                     /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     7803..7822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8041..8145)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(8223..8678)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8360..8381)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8572..8591
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8804..8
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8818..17
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggcccgacgt
       61 gatccatcga tgttaacacc ggtgacgtcg gatccaccgc catggggaga aaaaagattc
      121 agattacgag gataatggat gagcgtaaca gacaggtgac ttttacgaag aggaaatttg
      181 gattgatgaa gaaggcttat gagctgagcg tgctgtgcga ctgtgagatt gcactgatca
      241 tcttcaacag caccaacaag ctgttccagt acgccagcac tgacatggat aaggtgttgc
      301 tcaagtacac cgagtacaac gagccgcacg agagccggac aaactcagac attgtggagg
      361 cattgaacaa gaaagaaaac aaaggctctg aaagccccga tcctgactcc tcttatgcac
      421 tcaccccacg cactgaagaa aaatacaaaa aaattaatga agaatttgat aatatgatca
      481 agagtcataa aattcctgct gttccacctc ccagctttga gatgccagtt accatcccag
      541 tgtccagcca taacagtttg gtgtacagca atcctgtcag cacactggga aaccccaatc
      601 ttctgccact ggcccacccg tctctgcaga ggaatagtat gtctcctggt gtaacacata
      661 gacctccaag tgcaggtaac acaggcggtc tgatgggcgg agatctgaca tccggtgcag
      721 gcaccagcgc agggaatgga tacggcaacc cccggaactc accaggcctg ctggtctcac
      781 ctggtaacct gaacaagaat atacaagcca aatctcctcc ccctatgaat ctaggaatga
      841 ataatcgtaa gccagatctc cgcgttctta tcccacctgg cagcaagaac acgatgccat
      901 cagtgaatca aaggataaat aactcccagt cggctcagtc attggctacc ccggtggttt
      961 ccgtagcaac tcctacttta ccaggacaag gaatgggagg atatccatca gccatttcaa
     1021 caacatatgg tactgagtac tctctgagta gcgcagatct gtcatctctg tctggcttca
     1081 acactgccag tgcgctccac ctcggctctg taactggctg gcagcagcag cacctacata
     1141 acatgccgcc atctgccctc agtcagttgg gagaccgtac caccacccct tcgagatacc
     1201 cacaacacac cacgcgccac gaggcgggga ggtctcctgt tgacagcttg agcagctgta
     1261 gcagttccta cgatgggagc gaccgagagg atcaccggaa cgaattccac tcccccattg
     1321 gactcaccag accttcgccg gacgaaaggg aaagtccttc agtcaagcgc atgcgactct
     1381 ctgaaggatg ggcaacagct agcggctccg gcgagggcag gggaagtctt ctaacatgcg
     1441 gggacgtgga ggaaaatccc ggcccaacta gtatggtgag caagggcgag gaggtcatca
     1501 aagagttcat gcgcttcaag gtgcgcatgg agggctccat gaacggccac gagttcgaga
     1561 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     1621 ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc atgtacggct
     1681 ccaaggcgta cgtgaagcac cccgccgaca tccccgatta caagaagctg tccttccccg
     1741 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggtctggtg accgtgaccc
     1801 aggactcctc cctgcaggac ggcacgctga tctacaaggt gaagatgcgc ggcaccaact
     1861 tcccccccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tccaccgagc
     1921 gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca ccaggccctg aagctgaagg
     1981 acggcggcca ctacctggtg gagttcaaga ccatctacat ggccaagaag cccgtgcaac
     2041 tgcccggcta ctactacgtg gacaccaagc tggacatcac ctcccacaac gaggactaca
     2101 ccatcgtgga acagtacgag cgctccgagg gccgccacca cctgttcctg gggcatggca
     2161 ccggcagcac cggcagcggc agctccggca ccgcctcctc cgaggacaac aacatggccg
     2221 tcatcaaaga gttcatgcgc ttcaaggtgc gcatggaggg ctccatgaac ggccacgagt
     2281 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     2341 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtccccc cagttcatgt
     2401 acggctccaa ggcgtacgtg aagcaccccg ccgacatccc cgattacaag aagctgtcct
     2461 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggt ctggtgaccg
     2521 tgacccagga ctcctccctg caggacggca cgctgatcta caaggtgaag atgcgcggca
     2581 ccaacttccc ccccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcca
     2641 ccgagcgcct gtacccccgc gacggcgtgc tgaagggcga gatccaccag gccctgaagc
     2701 tgaaggacgg cggccgctac ctggtggagt tcaagaccat ctacatggcc aagaagcccg
     2761 tgcaactgcc cggctactac tacgtggaca ccaagctgga catcacctcc cacaacgagg
     2821 actacaccat cgtggaacag tacgagcgct ccgagggccg ccaccacctg ttcctgtacg
     2881 gcatggacga gctgtacaag aagcttggca gcggcgccac aaacttctct ctgctaaagc
     2941 aagcaggtga tgttgaagaa aaccccgggc ctctcgagat gtaccaaagc ctggccatgg
     3001 ccgccaacca cggccccccg cccggcgcct acgaagcagg tggccctggc gccttcatgc
     3061 acagcgcggg cgccgcgtcc tcgcccgtct acgtgcccac tccgcgggtg ccgtcctctg
     3121 tgctgggcct gtcctacctg cagggcggtg gcagtgccgc tgcagctgga accacctcgg
     3181 gtggcagctc cggggccggc ccgtcgggtg cagggcctgg gacccagcag ggtagccctg
     3241 gctggagcca agctggagcc gagggagccg cctacacccc gccgcccgtg tccccgcgct
     3301 tctctttccc ggggactact gggtccctgg cagcagctgc cgccgctgcc gcagcccggg
     3361 aagctgcagc ctacggcagt ggcggcgggg cggcgggcgc tggtctggct ggccgagagc
     3421 agtacgggcg tccgggcttc gccggctcct actccagccc ctacccagcc tacatggccg
     3481 acgtgggagc atcctgggcc gcagccgctg ccgcctctgc cggccccttc gacagcccag
     3541 tcctgcacag cctgcctgga cgggccaacc ctggaagaca ccccaatctc gatatgtttg
     3601 atgacttctc agaaggcaga gagtgtgtca attgtggggc catgtccacc ccactctgga
     3661 ggcgagatgg gacgggacac tacctgtgca atgcctgtgg cctctatcac aagatgaacg
     3721 gcatcaaccg gcccctcatt aagcctcagc gccgcctgtc cgcttcccgc cgggtaggcc
     3781 tctcctgtgc caactgccag actaccacca ccacgctgtg gcgtcgtaat gccgagggtg
     3841 agcctgtatg taatgcctgc ggcctctaca tgaagctcca tggggttccc aggcctcttg
     3901 caatgcggaa ggaggggatt caaaccagaa aacggaagcc caagaacctg aataaatcta
     3961 agacgccagc aggtcctgct ggtgagaccc tccctccctc cagtggtgcc tccagcggta
     4021 actccagcaa tgccactagc agcagcagca gcagtgaaga gatgcgcccc atcaagacag
     4081 agcccgggct gtcatctcac tatgggcaca gcagctccat gtcccagaca ttcagtactg
     4141 tgtccggcca cgggccctcc atccatccag tgctgtctgc tctgaagctg tccccacaag
     4201 gctatgcatc tcctgtcact cagacatcgc aggccagctc caagcaggac tcttggaaca
     4261 gcctggtcct ggctgacagt catggggaca taatcaccgc gtccggaggc tcgggccagt
     4321 gtactaatta tgctctcttg aaattggctg gagatgttga gagcaaccca ggtccctcta
     4381 gaatggccga tacagatgag ggctttggcc tggcgcgcac gcctctggag cctgattcca
     4441 aagacaggtc ttgcgattcg aaacctgaga gtgctctggg ggctcccagc aagtctccat
     4501 catccccgca ggctgccttc acccagcagg gcatggaagg aatcaaggtg tttcttcatg
     4561 aacgtgaact gtggctgaag ttccacgaag tgggcacaga gatgatcatc accaaggcag
     4621 ggaggagaat gtttcctagt tacaaagtga aggtgactgg ccttaatccc aaaacgaagt
     4681 atattcttct catggatatt gttcccgcag acgaccacag atataaattt gctgataaca
     4741 aatggtccgt aactggcaaa gcagagcctg ccatgccggg gcgcctttac gtgcacccgg
     4801 actccccagc aaccggagcc cactggatgc gacaacttgt ctccttccag aagctcaaac
     4861 tcaccaacaa ccacctggac ccgtttggac acattatcct gaactccatg cacaaatacc
     4921 agccccgatt acacatcgtg aaagcagacg aaaataatgg gttcggttca aagaacactg
     4981 cgttttgcac ccacgtcttc ccggagacag cttttatcgc tgtgacttcg taccagaatc
     5041 acaagatcac acagctgaaa attgagaaca accccttcgc caaaggcttt cggggcagtg
     5101 atgacctgga gttacacagg atgtctcgga tgcaaagtaa agagtatcct gtggttccca
     5161 ggagcacagt gaggcacaaa gtcacctcca accacagccc cttcagcagc gagacccgag
     5221 ctctctccac ctcatccaat ttagggtccc agtaccagtg tgagaatggt gtctctggcc
     5281 cctcccagga ccttctgccc ccacctaacc catacccact ggcccaggag cacagccaaa
     5341 tttaccactg taccaagagg aaagatgagg aatgttccag cacggagcac ccctataaga
     5401 agccgtacat ggagacatcc cccagcgagg aagacacctt ctatcgctcg ggctaccccc
     5461 agcagcaggg cctgagtacc tcttacagga cagagtcggc ccagcggcag gcctgcatgt
     5521 atgccagctc cgctcccccc agcgagcccg tgcctagcct ggaggacatc agctgtaaca
     5581 catggcccag catgccctcc tatagcagct gtaccgtcac caccgtgcag cccatggacc
     5641 gtcttcccta ccagcacttc tccgctcatt tcacctcggg gcccctggtc cctcggttgg
     5701 ctggcatggc caaccatggt tctccccagc tcggcgaagg gatgtttcag caccagacct
     5761 cagtggccca tcagcctgtg gtcaggcagt gcgggcctca gactggcctt cagtctccgg
     5821 gcggcctcca gcccccagag tttctctaca ctcacggcgt gcccaggacc ctgtcccccc
     5881 atcagtatca ctcggtacac ggcgtcggca tggtgccaga gtggagtgag aatagctaag
     5941 tcgacgcggc cgcccaacgc gttggatgca tagcttgagt attctatagt gtcacctaaa
     6001 tagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     6061 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     6121 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6181 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6241 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6301 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6361 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6421 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6481 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6541 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6601 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6661 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6721 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6781 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6841 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6901 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6961 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7021 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7081 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7141 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7201 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7261 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7321 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7381 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7441 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7501 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7561 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7621 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7681 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7741 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7801 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7861 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7921 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7981 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8041 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8101 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8161 agtgccacct gatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     8221 ggaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     8281 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     8341 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     8401 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     8461 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     8521 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     8581 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     8641 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc attcgccatt caggctgcgc
     8701 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     8761 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgtt
//
