LOCUS       Exported                4816 bp ds-DNA     circular SYN 02-DEC-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4816)
  AUTHORS   AG Stein
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Dec 2, 2019 from SnapGene Viewer 5.0.6
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4816
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(15..36)
                     /label=Amp res start rv
     primer_bind     complement(128..147)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     744..767
                     /label=Amp res end fw
     primer_bind     858..885
                     /label=front of ori fw
     primer_bind     complement(890..910)
                     /label=front of ori rv
     rep_origin      936..1524
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1425..1444
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1537..1558
                     /label=control fw on bb
     primer_bind     complement(1565..1586)
                     /label=amp bb gblock2 rev
     primer_bind     1631..1660
                     /label=Front of pT7 mfe1
     primer_bind     1638..1660
                     /label=pET24-front of pT7_fw
     promoter        1744..1766
                     /label=pT7
                     /note="Promotor T7"
     promoter        1744..1762
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           1767..1811
     primer_bind     complement(1803..1817)
                     /label=UTR Met Btsa1 rv
     primer_bind     1817..1839
                     /label=EcoRI for MBP
     CDS             1818..2915
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(1857..1892)
                     /label=mbp bsa1 mut rv
     primer_bind     1863..1898
                     /label=mbp bsa1 mut fw
     CDS             2928..2948
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2955..2978
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     misc_feature    2988..3011
                     /label=Autoinhibitory domain TVMV
     CDS             3014
                     /codon_start=2
                     /label=linker
                     /translation=""
     primer_bind     3018..3042
                     /label=FKBP fw Bbs1
     misc_feature    3019
                     /label=cutting site 1 1
     CDS             3021..3338
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="VQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPF
                     KFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLK
                     LE"
     primer_bind     complement(3314..3340)
                     /label=FKBP rv BsrD1
     primer_bind     3363..3390
                     /label=FRB fw Bbs1
     CDS             3363..3364
                     /codon_start=1
                     /label=short ggs linker
                     /translation=""
     CDS             3366..3638
                     /codon_start=1
                     /product=" FRB Stein 2014 shortened (-VA)"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRI"
     primer_bind     3366..3387
                     /label=start FRB Mfe1
     primer_bind     complement(3620..3640)
                     /label=FRB rv BsrD1
     CDS             3645..3646
                     /codon_start=1
                     /label=short ggs linker
                     /translation=""
     CDS             3648..4313
                     /codon_start=1
                     /label=TVMV
                     /translation="SKALLKGVRDFNPISACVCLLENSSDGHSERLFGIGFGPYIIANQ
                     HLFRRNNGELTIKTMHGEFKVKNSTQLQMKPVEGRDIIVIKMAKDFPPFPQKLKFRQPT
                     IKDRVCMVSTNFQQKSVSSLVSESSHIVHKEDTSFWQHWITTKDGQCGSPLVSIIDGNI
                     LGIHSLTHTTNGSNYFVEFPEKFVATYLDAADGWCKNWKFNADKISWGSFTLVEDAPED
                     "
     primer_bind     complement(4050..4083)
                     /label=mutTVMV C151A rv
     primer_bind     4084..4117
                     /label=mutTVMV C151A fw
     primer_bind     4314..4336
                     /note="color: black; sequence: GGCATTGCaTAAagccccgacaG"
     misc_feature    4323..4325
                     /label=final stop codon
     terminator      4371..4442
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4458..4485
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     4494..4516
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4529..4547)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        4621..4725
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4726..4816,1..770)
                     /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     join(4803..4816,1..12)
                     /label=amp bb gblock2 fw
ORIGIN
        1 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
       61 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
      121 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
      181 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
      241 ttacggatgg catgacagta agagaattat gtagtgctgc cataaccatg agtgataaca
      301 cagcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
      361 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
      421 taccaaacga cgagcgtgac accacgatgc ctgtagcgat ggcaacaacg ttgcgcaaac
      481 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
      541 cggataaagt tgcaggacca cttcttcgct cagcacttcc agctggttgg tttattgctg
      601 ataaatctgg agccggtgag cgtggatctc gcggtatcat agcagcactg gggccagatg
      661 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
      721 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
      781 aagtttactc atatatactt tgaattcaaa cttcattttt aatttaaaag gatctaggtg
      841 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
      901 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
      961 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1021 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1081 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1141 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1201 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1261 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1321 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1381 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1441 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     1501 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     1561 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     1621 cgtattaccg ccaattgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     1681 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattcta
     1741 gagtaatacg actcactata gggagaaata attttgttta actttaaaaa aaaaaagaag
     1801 gagaataatc tatggggaaa atcgaagaag gtaaactggt aatctggatt aacggcgata
     1861 aaggctataa cggtctggct gaagtcggta agaaattcga gaaagatacc ggaattaaag
     1921 tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg
     1981 atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc
     2041 tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg
     2101 atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc
     2161 tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc
     2221 tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt
     2281 acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca
     2341 agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc
     2401 tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag
     2461 ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca
     2521 tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat
     2581 ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc
     2641 tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata
     2701 aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg gtgaaagatc
     2761 cgcgtattgc cgccactatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc
     2821 agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc
     2881 agactgtcga tgaagccctg aaagacgcgc agactaattc gagctcggag aacctgtatt
     2941 tccagtccag tggctggagc cacccgcagt tcgaaaagtc aggaggtcgc gaatatgtgc
     3001 gctttgcgcc gggtccgggg gtgcaggtgg aaaccatttc tccaggcgat ggccgtacgt
     3061 tcccgaagcg cggtcaaaca tgcgtagtcc attacaccgg tatgctggaa gatggcaaaa
     3121 agttcgattc gtcccgtgac cgcaacaaac cctttaagtt catgttggga aaacaagaag
     3181 ttatccgtgg ctgggaggaa ggcgtagcgc aaatgtccgt tggacaacgt gcaaaattga
     3241 caatctctcc cgactacgcg tatggagcga ccggccatcc aggtatcatt ccacctcacg
     3301 ctacactggt gttcgatgtt gaactgctga aactggaagg tccgcctcca ccacctccgc
     3361 cagggattct gtggcacgag atgtggcatg agggcctgga agaggcatct cgtctgtatt
     3421 ttggtgaacg caacgttaaa ggtatgttcg aggttttgga gccacttcac gctatgatgg
     3481 agcgcggtcc acagaccttg aaagaaacct cgtttaatca agcgtatggc cgtgatctga
     3541 tggaggcaca ggagtggtgt cgtaagtaca tgaagagtgg gaacgtaaag gacctgactc
     3601 aggcgtggga tctgtattac cacgtgttcc gtcgcattgg tccggggtcc aaggcgcttc
     3661 tgaaaggcgt gcgtgacttc aatcctattt ctgcctgtgt atgtttgtta gagaacagtt
     3721 ctgacggcca ctctgaaagg ctattcggta taggcttcgg accgtacatc atcgccaacc
     3781 aacacctatt tagaagaaat aacggcgaac tgaccatcaa aaccatgcac ggtgagttca
     3841 aagttaagaa ctccactcag cttcagatga aacctgttga aggaagagat attatagtga
     3901 ttaagatggc aaaagacttt ccgccatttc cacaaaaatt gaaatttcgt cagccgacca
     3961 taaaagatag agtttgtatg gtaagtacta actttcaaca gaaatcagtc tcaagtcttg
     4021 ttagcgaaag tagtcacatt gtgcacaaag aggatactag cttttggcag cactggataa
     4081 ctacgaaaga cggtcagtgt ggtagtccat tggtatcaat tatagacgga aatatattag
     4141 gtatccacag tctaactcat actaccaatg gttccaatta tttcgtcgag tttcctgaaa
     4201 agtttgtggc gacttatctg gacgcagccg acggatggtg caaaaactgg aagtttaacg
     4261 cagacaaaat aagttgggga tcattcacct tagttgagga tgcaccggag gacggcattg
     4321 cataaagccc cgacagtaaa aagagaccct gactagccga ttccaggcat caaataaaac
     4381 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     4441 tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat actccgggag
     4501 ctgcatgtgt cagaggttta acgaaagggc ctcgtgatac gcctattttt ataggttaat
     4561 gtcatgataa taatggtttc ttagacgtca ggtggcaggc ttacttttcg gggaaatgtg
     4621 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4681 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4741 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4801 gaaacgctgg tgaaag
//
