LOCUS       Exported                6330 bp DNA     circular SYN 03-JUN-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6330)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Jul 21, 2021 from SnapGene Viewer 5.3.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6330
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     CDS             complement(119..1201)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(1202..1279)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1325..1344
                     /label=T7 promoter
     promoter        1325..1343
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1344..1368
                     /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     complement(1368..1422)
                     /label=MSNikJT Vector.REV
     primer_bind     1401..1451
                     /label=MSNikJTH Fragment 1.FOR
     RBS             1401..1405
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1412..2512
                     /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="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2369..2378
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     2486..2509
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     primer_bind     complement(2524..2568)
                     /label=MSNikJT Fragment 1.REV
     primer_bind     2554..2588
                     /label=MSNikJT Fragment 2.FOR
     CDS             2567..2857
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     2753..2775
                     /label=Sumo fwd
     CDS             2864..4225
                     /codon_start=1
                     /label=NikJ
                     /translation="MGEIVDEPRKILVIWPPQVLSYFNAGHHLALYQVTGHLRAVYPGA
                     QVTAIDASVERLTWKDLGSRLFQERYDVIAVMNEFDGIDGLRRLVSYARALNPDAPIVT
                     FGRLSGVNPRFFERFDIDAIVENGDFESGVVAAIEAFRGRAADAPGVHLRRDGSWAAPK
                     RRGDLLPAEDWYLPTPDEIPYGHYDALYFDDANKFCGIPRRRELVVPAARGCPVGCSYC
                     EVPLIYTRKERRLTVERTIQYIEESFAAAPFEYVAFYAPTFTLDKKWMEELCRELTGRG
                     SPYPWKCATTVHHLSEGMIRSMGAAGCVRISVGLETLDPQEGALLPRAKQKSAMDLDRL
                     SAWCKESGIELNCFVIVGLPGTTIEGAERTIEAVQERGGRARATVYAPWELAGPDLLDH
                     EMAAFNRQLFVPGTHALTDDELARGYDIVFGSQPDVTQVFENIPARAAGPALRPA"
     primer_bind     complement(4211..4233)
                     /label=MSNikJT Fragment 2.REV
     primer_bind     4219..4246
                     /label=MSNikJT Fragment 3.FOR
     CDS             4241..4261
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4274..4297
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4350..4368)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4364..4411
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4535..4554)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4703..4733)
                     /label=MSNikJT Fragment 3.REV
     primer_bind     4719..4750
                     /label=MSNikJT Vector.FOR
     promoter        4726..4828
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene"
     primer_bind     4726..4744
                     /label=SUMOCm F2.FOR
     CDS             4829..5488
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5482..5505
                     /label=SUMOCm V.FOR
     rep_origin      complement(5504..6253)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
ORIGIN
        1 catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
       61 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
      121 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      181 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
      241 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
      301 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
      361 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
      421 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
      481 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
      541 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
      601 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
      661 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      721 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      781 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      841 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      901 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      961 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     1021 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     1081 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     1141 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     1201 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     1261 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     1321 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tgtagaaata
     1381 attttgttta actttaataa ggagatatac catgaaaatc gaagaaggta aactggtaat
     1441 ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga aattcgagaa
     1501 agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca
     1561 ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
     1621 ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc aggacaagct
     1681 gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc
     1741 tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg
     1801 ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt
     1861 caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt
     1921 caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
     1981 agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga
     2041 ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc
     2101 gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac
     2161 cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc
     2221 cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg
     2281 tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
     2341 agagttggcg aaagatccac gtattgccgc caccatggaa aacgcccaga aaggtgaaat
     2401 catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa
     2461 cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga ctaattcgag
     2521 ctcgaacaac aacaacaata acaataacaa caacctcggg agcactagtg actcagaagt
     2581 caatcaagaa gctaagccag aggtcaagcc agaagtcaag cctgagactc acatcaattt
     2641 aaaggtgtcc gatggatctt cagaaatctt ctttaagatc aaaaagacca ctcctttaag
     2701 aaggctgatg gaagcgttcg ctaaaagaca gggtaaggaa atggactcct taagattctt
     2761 gtacgacggt attagaattc aagctgatca gacccctgaa gatttggaca tggaggataa
     2821 cgatattatt gaggctcaca gagaacagat tggtggatcc catatgggcg aaatcgtgga
     2881 cgaaccgcgt aaaatcctgg tcatctggcc gccgcaagtg ctgtcatact tcaatgctgg
     2941 tcatcacctg gcactgtatc aggtgaccgg tcatctgcgt gctgtgtacc cgggtgcgca
     3001 agttaccgcc attgatgcat cagttgaacg tctgacgtgg aaagatctgg gttcgcgcct
     3061 gttccaggaa cgttatgatg tgattgccgt tatgaatgaa tttgatggca tcgacggtct
     3121 gcgtcgcctg gttagctacg cccgtgcact gaacccggat gcaccgattg ttacctttgg
     3181 ccgtctgtct ggtgtcaatc cgcgcttttt cgaacgtttt gatattgacg ccatcgtgga
     3241 aaacggcgat ttcgaaagcg gtgtggttgc ggccatcgaa gcctttcgcg gccgtgcagc
     3301 tgatgcaccg ggtgttcatc tgcgtcgcga cggttcgtgg gcagcaccga aacgtcgcgg
     3361 tgatctgctg ccggcagaag actggtatct gccgaccccg gatgaaattc cgtatggtca
     3421 ctacgacgca ctgtactttg atgacgctaa caagttctgc ggtatcccgc gtcgccgtga
     3481 actggtcgtg ccggcagctc gtggttgtcc ggtcggttgc agctattgtg aagtgccgct
     3541 gatttacacg cgcaaagaac gccgtctgac cgttgaacgt acgattcagt atatcgaaga
     3601 atcttttgcg gccgcaccgt tcgaatatgt cgctttttac gcgccgacct tcacgctgga
     3661 taaaaagtgg atggaagaac tgtgccgcga actgaccggc cgtggttcac cgtatccgtg
     3721 gaagtgtgcg accacggttc atcacctgag cgagggtatg attcgctcta tgggcgctgc
     3781 gggttgcgtt cgtatcagtg tcggtctgga aacgctggat ccgcaggaag gcgcactgct
     3841 gccgcgtgca aaacaaaagt ccgctatgga tctggaccgt ctgagtgcct ggtgcaaaga
     3901 atccggtatt gaactgaatt gttttgttat cgtcggcctg ccgggtacca cgattgaagg
     3961 cgcggaacgc accatcgaag cagtgcagga acgtggcggt cgtgctcgtg caacggttta
     4021 tgcaccgtgg gaactggcag gtccggatct gctggaccat gaaatggccg catttaatcg
     4081 ccaactgttc gtcccgggca cccacgcact gacggatgac gaactggctc gtggctacga
     4141 tattgtgttt ggttcccagc cggacgtcac ccaagtgttc gaaaacatcc cggcgcgcgc
     4201 tgcgggcccg gccctgcgtc cggcacaggg aaacggtacc gaaaacctgt attttcaggg
     4261 ctcgacaatg gatcaccacc accaccacca ccaccactaa cctaggctgc taaacaaagc
     4321 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     4381 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     4441 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4501 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4561 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4621 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4681 agtgggccat cgccctgata gacggttttt cgccctttga cgttgtgatc ggcacgtaag
     4741 aggttccaac tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttatc
     4801 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt
     4861 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg
     4921 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa
     4981 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc
     5041 ggaatttcgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg
     5101 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga
     5161 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct
     5221 ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt
     5281 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt
     5341 caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
     5401 tcatcatgcc gtctgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta
     5461 ctgcgatgag tggcagggcg gggcgtaagc agcatgcagc gctcttccgc ttcctcgctc
     5521 actgactcgc tacgctcggt cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg
     5581 gtaatacggt tatccacaga atcaggggat aaagccggaa agaacatgtg agcaaaaagc
     5641 aaagcaccgg aagaagccaa cgccgcaggc gtttttccat aggctccgcc cccctgacga
     5701 gcatcacaaa aatcgacgct caagccagag gtggcgaaac ccgacaggac tataaagata
     5761 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5821 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5881 ttggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5941 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6001 acacgactta tcgccactgg cagcagccat tggtaactga tttagaggac tttgtcttga
     6061 agttatgcac ctgttaaggc taaactgaaa gaacagattt tggtgagtgc ggtcctccaa
     6121 cccacttacc ttggttcaaa gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg
     6181 tagcggtggt ttttctttat ttatgagatg atgaatcaat cggtctatca agtcaacgaa
     6241 cagctattcc gttactctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     6301 tcagccccat acgatataag ttgtaattct
//
