LOCUS       Exported                8124 bp DNA     circular SYN 27-MAR-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8124)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported Aug 4, 2021 from SnapGene Viewer 5.3.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8124
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        22..400
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    30..48
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    66..84
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    102..120
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    138..156
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    174..192
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    210..228
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    246..264
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    407..415
                     /label=Kozak
     CDS             413..1129
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(459..479)
                     /label=EGFP-N-primer-Reverse
     primer_bind     1066..1087
                     /label=EGFP-C-primer-Forward
     CDS             1175..1195
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1208..1252
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1271..1930
                     /codon_start=1
                     /label=hMeikin (155-373)
                     /translation="KSDYLDWECPNLEEHMQWKNSTLLDTSKAVAIEKAPQFSNVSAIF
                     STSSEDYQKCHRKTVMTVADQNVSPKAKCASNSESDNAACEILLAEKTCPSTPEKTKKK
                     KTNSSTPGKKNRGLLTSTPSSETAGFVIDLSSVQKASFEELFPNVSNYVNSNEIVPVSS
                     LQENSSNEFPANASEICCIIRTSPGTRQVKNKGVIVKKKKYSLPKDTPQDIIIKMA"
     primer_bind     complement(2232..2249)
                     /label=BGHRpA-primer-Reverse
     polyA_signal    2238..2462
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    2479..2600
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     2544..2563
                     /label=SV40pA-primer-Reverse
     CDS             complement(2631..3230)
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     CDS             complement(3237..3290)
                     /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"
     misc_feature    3296..3321
                     /label=SA
                     /note="splice acceptor site"
     misc_feature    3328..4131
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     rep_origin      complement(4151..4739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4910..5770)
                     /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"
     promoter        complement(5771..5875)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    5912..6748
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     polyA_site      6756..6811
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     CDS             complement(6840..7586)
                     /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"
     LTR             7609..7877
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     promoter        complement(7890..8101)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 tttcgtcttc aacaattctt cgagtttact ccctatcagt gatagagaac gtatgaagag
       61 tttactccct atcagtgata gagaacgtat gcagacttta ctccctatca gtgatagaga
      121 acgtataagg agtttactcc ctatcagtga tagagaacgt atatccagtt tactccctat
      181 cagtgataga gaacgtatct acagtttact ccctatcagt gatagagaac gtatatccag
      241 tttactccct atcagtgata gagaacgtat aagctttagg cgtgtacggt gggcgcctat
      301 aaaagcagag ctcgtttagt gaaccgtcag atcgcctgga gcaattccac aacacttttg
      361 tcttatacca actttccgta ccacttccta ccctcgtaaa gtcgacgcca ccatggtgag
      421 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
      481 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
      541 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
      601 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
      661 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
      721 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
      781 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
      841 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
      901 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
      961 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1021 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     1081 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ccggagggga
     1141 tcggtggagc tccaccggtg gcggccgctc tagagagaat ctttattttc agggcgccgc
     1201 caaattcaaa gaaaccgctg ctgctaaatt cgaacgccag cacatggaca gcggaggtgg
     1261 aggtctcgag aagtccgact acctggactg ggagtgcccc aatctggagg agcacatgca
     1321 gtggaagaac agcaccctgc tcgacacctc caaggccgtg gctatcgaga aggctcccca
     1381 gttcagcaac gtcagcgcca tcttctccac cagcagcgag gactaccaga agtgccacag
     1441 gaagaccgtg atgaccgtgg ccgatcagaa cgtgtccccc aaggccaagt gcgccagcaa
     1501 tagcgagagc gacaacgctg cctgtgaaat cctgctggcc gagaagacat gccccagcac
     1561 ccccgagaag accaagaaga agaagaccaa cagcagcaca cctggcaaga agaacagagg
     1621 actgctgacc tccacaccta gctccgagac cgccggcttt gtgatcgacc tgagctccgt
     1681 gcagaaggcc tcctttgagg agctgttccc caacgtgagc aactacgtga acagcaacga
     1741 gatcgtcccc gtgtccagcc tccaggagaa cagctccaac gagttccccg ccaacgcctc
     1801 cgagatctgc tgcatcatca ggacctcccc tggcaccagg caggtgaaaa acaagggcgt
     1861 gattgtgaag aagaaaaagt acagcctgcc caaggacacc ccccaggaca tcatcatcaa
     1921 gatggcctaa gaattcctta agtacgtacc gcgggctagc ggatccatgc atacatgtga
     1981 tatcactagt gattttaggg agagtttagt atccctggtt cagaactcca gaaaactaca
     2041 gaacattaga gacaatgtgg aaaaggaatc gaaacgactg aaaatatctt aacccgggat
     2101 ccaccggatc tagataactg atcataatca gccataccac atttgtagag gttttacttg
     2161 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattaaac
     2221 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2281 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2341 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2401 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2461 ggggtaccac agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     2521 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     2581 taagctgcaa taaacaagtt ggggtgggcg aagaactcca gcatacgcgt tcaggcaccg
     2641 ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc ggcggtgacg
     2701 gtgaagccga gccgctcgta gaaggggagg ttgcggggcg cggaggtctc caggaaggcg
     2761 ggcaccccgg cgcgctcggc cgcctccact ccggggagca cgacggcgct gcccagaccc
     2821 ttgccctggt ggtcgggcga gacgccgacg gtggccagga accacgcggg ctccttgggc
     2881 cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga accgctcaac
     2941 tcggccatgc gcgggccgat ctcggcgaac accgcccccg cttcgacgct ctccggcgtg
     3001 gtccagaccg ccactgcggc gccgtcgtcc gcgacccaca ccttgccgat gtcgagcccg
     3061 acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg gtccggatcg
     3121 acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg tacggccctg
     3181 gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt actcggtcat atcgataggg
     3241 ccgggattct cctccacgtc accgcatgtt agaagacttc ctctgccctc tccgccctgt
     3301 gggaggaaga gaagaggtca ggcgcgtgcc ccactgtggg gtggagggga cagataaaag
     3361 tacccagaac cagagccaca ttaaccggcc ctgggaatat aaggtggtcc cagctcgggg
     3421 acacaggatc cctggaggca gcaaacatgc tgtcctgaag tggacatagg ggcccgggtt
     3481 ggaggaagaa gactagctga gctctcggac ccctggaaga tgccatgaca gggggctgga
     3541 agagctagca cagactagag aggtaagggg ggtaggggag ctgcccaaat gaaaggagtg
     3601 agaggtgacc cgaatccaca ggagaacggg gtgtccaggc aaagaaagca agaggatgga
     3661 gaggtggcta aagccaggga gacggggtac tttggggttg tccagaaaaa cggtgatgat
     3721 gcaggcctac aagaagggga ggcgggacgc aagggagaca tccgtcggag aaggccatcc
     3781 taagaaacga gagatggcac aggccccaga aggagaagga aaagggaacc cagcgagtga
     3841 agacggcatg gggttgggtg agggaggaga gatgcccgga gaggacccag acacggggag
     3901 gatccgctca gaggacatca cgtggtgcag cgccgagaag gaagtgctcc ggaaagagca
     3961 tccttgggca gcaacacagc agagagcaag gggaagaggg agtggaggaa gacggaacct
     4021 gaaggaggcg gcagggaagg atctgggcca gccgtagagg tgacccaggc cacaagctgc
     4081 agacagaaag cggcacaggc ccaggggaga gaatgctggt cagagaaagc aggcgcgcct
     4141 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4201 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4261 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4321 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4381 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4441 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4501 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4561 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4621 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4681 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4741 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4801 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4861 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4921 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4981 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5041 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5101 gaagggcaga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5161 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5221 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5281 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5341 tcggtcctcc aatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5401 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5461 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5521 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5581 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5641 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5701 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5761 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5821 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5881 ttccccgaaa agtgcttaag cccttgccct tagagcagag ccaggaaccc ctgtagggaa
     5941 ggggcaggag agccaggggc atgagatggt ggacgaggaa gggggacagg gaagcctgag
     6001 cgcctctcct gggcttgcca aggactcaaa cccagaagcc cagagcaggg ccttagggaa
     6061 gcgggaccct gctctgggcg gaggaatatg tcccagatag cactggggac tctttaagga
     6121 aagaaggatg gagaaagaga aagggagtag aggcggccac gacctggtga acacctagga
     6181 cgcaccattc tcacaaaggg agttttccac acggacaccc ccctcctcac cacagccctg
     6241 ccaggacggg gctggctact ggccttatct cacaggtaaa actgacgcac ggaggaacaa
     6301 tataaattgg ggactagaaa ggtgaagagc caaagttaga actcaggacc aacttattct
     6361 gattttgttt ttccaaactg cttctcctct tgggaagtgt aaggaagctg cagcaccagg
     6421 atcagtgaaa cgcaccagac agccgcgtca gagcagctca ggttctggga gagggtagcg
     6481 cagggtggcc actgagaacc gggcaggtca cgcatccccc ccttccctcc caccccctgc
     6541 caagctctcc ctcccaggat cctctctggc tccatcgtaa gcaaacctta gaggttctgg
     6601 caaggagaga gatggctcca ggaaatgggg gtgtgtcacc agataaggaa tctgcctaac
     6661 aggaggtggg ggttagaccc aatatcagga gactaggaag gaggaggcct aaggatgggg
     6721 cttttctgtc accaatcctg tccctagtaa gagcttgaga gacacaaaaa attccaacac
     6781 actattgcaa tgaaaataaa tttcctttat tccggaataa aggaaattta gtacccgggt
     6841 taccctggga gcatgtcaag gtcaaaatcg tcaagagcgt cagcaggcag catatcaagg
     6901 tcaaagtcgt caagggcatc ggctgggagc atgtctaagt caaaatcgtc aagggcgtcg
     6961 gtcggcccgc cgctttcgca ctttagctgt ttctccaggc cacatatgat tagttccagg
     7021 ccgaaaagga aggcaggttc ggctccctgc cggtcgaaca gctcaattgc ttgtttcaga
     7081 agtgggggca tagaatcggt ggtaggtgtc tctctttcct cttttgctac ttgatgctcc
     7141 tgttcctcca atacgcagcc cagtgtaaag tggcccacgg cggacagagc gtacagtgcg
     7201 ttctccaggg agaagccttg ctgacacagg aacgcgagct gattttccag ggtttcgtac
     7261 tgtttctctg ttgggcgggt gccgagatgc actttagccc cgtcgcgatg tgagaggaga
     7321 gcacagcggt atgacttggc gttgttccgc agaaagtctt gccatgactc gccttccagg
     7381 gggcaggagt gggtatgatg cctgtccagc atctcgattg gcagggcatc gagcagggcc
     7441 cgcttgttct tcacgtgcca gtacagggta ggctgctcaa ctcccagctt ttgagcgagt
     7501 ttccttgtcg tcaggccttc gataccgact ccattgagta attccagagc agagtttatg
     7561 actttgctct tgtccagtct agacatggtg cttaattaac acagcttgga tctggaacgg
     7621 cgcagaacag aaaacgaaac aaagacgtag agttgagcaa gcagggtcag gcaaagcgtg
     7681 gagagccggc tgagtctagg taggctccaa gggagcgccg gacaaaggcc cggtctcgac
     7741 ctgagcttta aacttaccta gacggcggac gcagttcagg aggcacctca ggcgggaggc
     7801 ggcagaacgc gactcaaccg gcgtggatgg cggcctcagg tagggcggcg ggcgcgtgaa
     7861 ggagagatgc gagcccctcg aagcttcagc tgtgttctgg cggcaaaccc gttgcgaaaa
     7921 agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg
     7981 cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccc
     8041 gttcaattgc cgacccctcc ccccaacttc tcggggactg tgggcgatgt gcgctctgcc
     8101 cactgacggg caccggagag atct
//
