LOCUS       Exported                7714 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  PiggybacTransposon-based tunable and temporal expression control of 
            SARS-CoV2 N and mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    XLone-BSD SARS-CoV2 N P2A mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7714)
  TITLE     Inducible COVID19 SARS-CoV2 Nucleocapsid Expressing Plasmid in 
            Mammalian Cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7714)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7714
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..368
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    9..27
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    45..63
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    81..99
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    117..135
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    153..171
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    189..207
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    225..243
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1671..1727
                     /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             1731..1754
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1755..2462
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1897..1915)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2165..2184
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2668..2802
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   3078..3112
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3123..3140)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3123..3139)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3132..3154)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3348..3367)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3467..3489
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3527..3545)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3613..3717
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3718..4578
                     /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(3936..3955)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4749..5337
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5238..5257
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5491..5508
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5625..5646
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5661..5691
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5699..5715
                     /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     5704..5726
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5723..5739
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5723..5739
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5765..5827
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    6003..6084
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6201..6599)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(6600..6656)
                     /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             complement(6666..7409)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(7410..7433)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7433..7442
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     promoter        complement(7462..7673)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag
       61 agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga gtttactccc
      121 tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta
      181 cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag
      241 agaacgtata agctttgctt atgtaaacca gggcgcctat aaaagagtgc tgattttttg
      301 agtaaacttc aattccacaa cacttttgtc ttataccaac tttccgtacc acttcctacc
      361 ctcgtaaagg taccgccacc atgagcgata acggccccca aaaccaacgg aatgcccctc
      421 ggataacatt tggtggccct agtgattcca caggaagtaa ccaaaacgga gaacggtctg
      481 gagcccgctc taaacagagg cgaccacagg gattgccaaa taacacagcg tcatggttta
      541 cagccctgac tcaacatgga aaggaggact tgaaatttcc acgaggacag ggtgtaccaa
      601 taaacacaaa cagtagtcca gacgatcaga tcgggtatta tagaagggcc acgagacgca
      661 tcagaggtgg agatggtaaa atgaaagatc tcagtccgcg ctggtacttt tattatttgg
      721 gaaccggccc agaagcgggc ctcccatatg gagccaataa ggacgggatt atatgggtgg
      781 ctacagaggg agccctcaat acgccaaaag atcacatcgg aacaagaaac cctgctaata
      841 atgccgcaat cgtgctgcag ttgcctcagg ggacgacgct tcctaaaggc ttttacgcag
      901 aaggatcacg cggcggcagc caagcatcct caaggtctag ttccagaagt cgaaacagct
      961 ccagaaactc cacaccaggg tccagtaggg gcacaagtcc ggcgcggatg gcgggcaacg
     1021 gcggagacgc cgcactcgct ctgttgctcc tggaccggct caaccaactt gaatccaaga
     1081 tgagtggtaa gggacagcag caacaaggtc aaaccgtaac caagaaaagc gctgcagaag
     1141 cttctaaaaa acctcgacaa aaacggaccg ctacgaaggc atataacgtt actcaagcct
     1201 tcgggagacg agggccggag caaacccagg ggaattttgg agatcaggag ctcatccggc
     1261 aagggacaga ttacaaacat tggcctcaaa tcgctcagtt cgcgccgtcc gcaagtgctt
     1321 ttttcggaat gtctcgcatc ggtatggaag taacaccatc cgggacatgg ctcacttata
     1381 ccggcgccat caagctggac gacaaagatc caaatttcaa agatcaggtg attctcttga
     1441 ataaacacat agacgcgtat aagacgtttc cacccaccga acccaagaag gacaaaaaga
     1501 aaaaagctga tgaaacacaa gcgttgcccc agcgacagaa gaagcagcaa accgtcaccc
     1561 tcctgccagc cgctgatctc gatgacttct ccaagcaact tcaacagtcc atgagctcag
     1621 ccgattctac tcaggcgctc gaaggcggcg ggggagctag cggcagcggc gccactaact
     1681 tctccctgtt gaaacaagca ggggatgtcg aagagaatcc cgggccaatg gactacaagg
     1741 acgacgatga caaggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
     1801 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     1861 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     1921 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     1981 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     2041 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     2101 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     2161 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     2221 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     2281 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     2341 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     2401 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     2461 aagaattccg atcatattca ataacccact agtagaccac ctcccctgcg agctaagctg
     2521 gacagccaat gacgggtaag agagtgacat ttttcactaa cctaagacag gagggccgtc
     2581 agagctactg cctaatccaa agacgggtaa aagtgataaa aatgtatcac tccaacctaa
     2641 gacaggcgca gcttccgagg gatttgagat ccagacatga taagatacat tgatgagttt
     2701 ggacaaacca aaactagaat gcagtgaaaa aaatgcctta tttgtgaaat ttgtgatgct
     2761 attgccttat ttgtaaccat tataagctgc aataaacaag tttgatatct ataacaagaa
     2821 aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta
     2881 tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc
     2941 gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca
     3001 agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga
     3061 gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaagaat
     3121 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     3181 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     3241 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     3301 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     3361 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3421 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3481 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     3541 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     3601 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3661 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3721 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3781 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3841 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3901 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3961 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4021 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4081 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4141 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4201 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4261 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4321 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4381 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4441 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4501 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4561 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4621 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4681 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4741 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4801 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4861 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4921 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4981 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5041 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5101 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5161 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5221 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5281 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5341 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5401 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5461 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5521 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     5581 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     5641 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     5701 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caaggtcgac
     5761 ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg
     5821 acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa
     5881 gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt
     5941 tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt
     6001 tagcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     6061 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     6121 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct
     6181 gattatgatc ctctggagat ttagccctcc cacacataac cagagggcag caattcacga
     6241 atcccaactg ccgtcggctg tccatcactg tccttcacta tggctttgat cccaggatgc
     6301 agatcgagaa gcacctgtcg gcaccgtccg caggggctca agatgcccct gttctcattt
     6361 ccgatcgcga cgatacaagt caggttgcca gctgccgcag cagcagcagt gcccagcacc
     6421 acgagttctg cacaaggtcc cccagtaaaa tgatatacat tgacaccagt gaagatgcgg
     6481 ccgtcgctag agagagctgc gctggcgacg ctgtagtctt cagagatggg gatgctgttg
     6541 attgtagccg ttgctctttc aatgagggtg gattcttctt gagacaaagg cttggccatg
     6601 gggccggggt tctcctccac gtcgccggcc tgcttcagca ggctgaagtt ggtggcgccg
     6661 ctgcccccgg ggagcatgtc aaggtcaaaa tcgtcaagag cgtcagcagg cagcatatca
     6721 aggtcaaagt cgtcaagggc atcggctggg agcatgtcta agtcaaaatc gtcaagggcg
     6781 tcggtcggcc cgccgctttc gcactttagc tgtttctcca ggccacatat gattagttcc
     6841 aggccgaaaa ggaaggcagg ttcggctccc tgccggtcga acagctcaat tgcttgtttc
     6901 agaagtgggg gcatagaatc ggtggtaggt gtctctcttt cctcttttgc tacttgatgc
     6961 tcctgttcct ccaatacgca gcccagtgta aagtggccca cggcggacag agcgtacagt
     7021 gcgttctcca gggagaagcc ttgctgacac aggaacgcga gctgattttc cagggtttcg
     7081 tactgtttct ctgttgggcg ggtgccgaga tgcactttag ccccgtcgcg atgtgagagg
     7141 agagcacagc ggtatgactt ggcgttgttc cgcagaaagt cttgccatga ctcgccttcc
     7201 agggggcagg agtgggtatg atgcctgtcc agcatctcga ttggcagggc atcgagcagg
     7261 gcccgcttgt tcttcacgtg ccagtacagg gtaggctgct caactcccag cttttgagcg
     7321 agtttccttg tcgtcaggcc ttcgataccg actccattga gtaattccag agcagagttt
     7381 atgactttgc tcttgtccag tctagacatc ttatcgtcat cgtctttgta atccatggtg
     7441 gcggatcccg cgtcacgaca cctgtgttct ggcggcaaac ccgttgcgaa aaagaacgtt
     7501 cacggcgact actgcactta tatacggttc tcccccaccc tcgggaaaaa ggcggagcca
     7561 gtacacgaca tcactttccc agtttacccc gcgccacctt ctctaggcac cggttcaatt
     7621 gccgacccct ccccccaact tctcggggac tgtgggcgat gtgcgctctg cccactgacg
     7681 ggcaccggag ccactcgagt ggaattatca cctc
//
