LOCUS       40924_39808        6787 bp DNA     circular SYN 13-MAY-2021
DEFINITION  bacterial expression of hyperactive Tn5 transposase carrying E54K 
            and L372P mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6787)
  TITLE     Linnarsson lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6787)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6787)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6787
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=lac operator
                     /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)."
     RBS             59..81
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             155..1582
                     /codon_start=1
                     /label=Tn5 transposase
                     /note="transposase from the bacterial Tn5 transposon
                     (Reznikoff, 1993)"
                     /translation="MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKS
                     ITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYR
                     HQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESG
                     KWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGL
                     YLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNA
                     VLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAER
                     QRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTP
                     DECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQS
                     KLDGFLAAKDLMAQGIKI"
     CDS             1648..1665
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      1732..1779
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1816..2271
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2367..3179)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      3301..3889
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4043..4060
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    complement(4075..4217)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4303..4325
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4322..4510)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5285..5306)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5322..6401)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6402..6479)
                     /label=lacI promoter
     primer_bind     6685..6704
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatgatt accagtgcac tgcatcgtgc
      181 ggcggattgg gcgaaaagcg tgttttctag tgctgcgctg ggtgatccgc gtcgtaccgc
      241 gcgtctggtg aatgttgcgg cgcaactggc caaatatagc ggcaaaagca ttaccattag
      301 cagcgaaggc agcaaagcca tgcaggaagg cgcgtatcgt tttattcgta atccgaacgt
      361 gagcgcggaa gcgattcgta aagcgggtgc catgcagacc gtgaaactgg cccaggaatt
      421 tccggaactg ctggcaattg aagataccac ctctctgagc tatcgtcatc aggtggcgga
      481 agaactgggc aaactgggta gcattcagga taaaagccgt ggttggtggg tgcatagcgt
      541 gctgctgctg gaagcgacca cctttcgtac cgtgggcctg ctgcatcaag aatggtggat
      601 gcgtccggat gatccggcgg atgcggatga aaaagaaagc ggcaaatggc tggccgctgc
      661 tgcaacttcg cgtctgagaa tgggcagcat gatgagcaac gtgattgcgg tgtgcgatcg
      721 tgaagcggat attcatgcgt atctgcaaga taaactggcc cataacgaac gttttgtggt
      781 gcgtagcaaa catccgcgta aagatgtgga aagcggcctg tatctgtatg atcacctgaa
      841 aaaccagccg gaactgggcg gctatcagat tagcattccg cagaaaggcg tggtggataa
      901 acgtggcaaa cgtaaaaacc gtccggcgcg taaagcgagc ctgagcctgc gtagcggccg
      961 tattaccctg aaacagggca acattaccct gaacgcggtg ctggccgaag aaattaatcc
     1021 gccgaaaggc gaaaccccgc tgaaatggct gctgctgacc agcgagccgg tggaaagtct
     1081 ggcccaagcg ctgcgtgtga ttgatattta tacccatcgt tggcgcattg aagaatttca
     1141 caaagcgtgg aaaacgggtg cgggtgcgga acgtcagcgt atggaagaac cggataacct
     1201 ggaacgtatg gtgagcattc tgagctttgt ggcggtgcgt ctgctgcaac tgcgtgaatc
     1261 ttttactccg ccgcaagcac tgcgtgcgca gggcctgctg aaagaagcgg aacacgttga
     1321 aagccagagc gcggaaaccg tgctgacccc ggatgaatgc caactgctgg gctatctgga
     1381 taaaggcaaa cgcaaacgca aagaaaaagc gggcagcctg caatgggcgt atatggcgat
     1441 tgcgcgtctg ggcggcttta tggatagcaa acgtaccggc attgcgagct ggggtgcgct
     1501 gtgggaaggt tgggaagcgc tgcaaagcaa actggatggc tttctggccg cgaaagacct
     1561 gatggcgcag ggcattaaaa tctgacagta aaggtggata cggatccgaa ttcgagctcc
     1621 gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
     1681 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1741 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1801 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     1861 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     1921 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1981 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2041 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2101 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2161 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2221 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca
     2281 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     2341 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     2401 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     2461 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     2521 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     2581 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     2641 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     2701 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     2761 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     2821 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     2881 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     2941 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     3001 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     3061 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     3121 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     3181 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     3241 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3301 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3361 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3421 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3481 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3541 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3601 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3661 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3721 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3781 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3841 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3901 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3961 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4021 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     4081 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc
     4141 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     4201 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4261 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4321 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     4381 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     4441 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     4501 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     4561 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     4621 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     4681 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     4741 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     4801 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     4861 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     4921 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     4981 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     5041 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     5101 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     5161 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     5221 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg
     5281 cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5341 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5401 gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc
     5461 ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg
     5521 cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg
     5581 tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc
     5641 attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca
     5701 ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc
     5761 gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc
     5821 gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc
     5881 agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt
     5941 gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca
     6001 atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga
     6061 agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc
     6121 acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg
     6181 tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt
     6241 tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc
     6301 gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca
     6361 ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga
     6421 ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc
     6481 gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     6541 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     6601 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     6661 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     6721 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     6781 gcgaaat
//
