LOCUS       sequence-418885-        6783 bp    DNA     circular SYN 15-DEC-2025
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 6783)
  TITLE     Linnarsson lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6783)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: psfTn5
FEATURES             Location/Qualifiers
     source          1..6783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             5..27
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             38..55
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             80..100
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             101..1528
                     /codon_start=1
                     /product="transposase from the bacterial Tn5 transposon 
                     (Reznikoff, 1993)"
                     /label=Tn5 transposase
                     /translation="MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKS
                     ITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYR
                     HQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESG
                     KWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGL
                     YLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNA
                     VLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAER
                     QRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTP
                     DECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQS
                     KLDGFLAAKDLMAQGIKI"
     CDS             1594..1611
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1664..1682)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1678..1725
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1762..2217
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1849..1868)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2059..2080
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2309..3124)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2309..3124)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3032..3051
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3116..3135
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3246..3834
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3735..3754
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3988..4005
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4020..4159
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4085..4104)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4245..4267
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4261..4452)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5261..6343)
                     /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"
     primer_bind     6305..6324
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6344..6421)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6627..6646
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6730..6749
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6730..6748
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6749..6773
                     /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)."
ORIGIN
        1 taattttgtt taactttaag aaggagatat acatatgcac catcatcatc atcattcttc
       61 tggtgtagat ctgggtaccg agaacctgta cttccaatcc atgattacca gtgcactgca
      121 tcgtgcggcg gattgggcga aaagcgtgtt ttctagtgct gcgctgggtg atccgcgtcg
      181 taccgcgcgt ctggtgaatg ttgcggcgca actggccaaa tatagcggca aaagcattac
      241 cattagcagc gaaggcagca aagccatgca ggaaggcgcg tatcgtttta ttcgtaatcc
      301 gaacgtgagc gcggaagcga ttcgtaaagc gggtgccatg cagaccgtga aactggccca
      361 ggaatttccg gaactgctgg caattgaaga taccacctct ctgagctatc gtcatcaggt
      421 ggcggaagaa ctgggcaaac tgggtagcat tcaggataaa agccgtggtt ggtgggtgca
      481 tagcgtgctg ctgctggaag cgaccacctt tcgtaccgtg ggcctgctgc atcaagaatg
      541 gtggatgcgt ccggatgatc cggcggatgc ggatgaaaaa gaaagcggca aatggctggc
      601 cgctgctgca acttcgcgtc tgagaatggg cagcatgatg agcaacgtga ttgcggtgtg
      661 cgatcgtgaa gcggatattc atgcgtatct gcaagataaa ctggcccata acgaacgttt
      721 tgtggtgcgt agcaaacatc cgcgtaaaga tgtggaaagc ggcctgtatc tgtatgatca
      781 cctgaaaaac cagccggaac tgggcggcta tcagattagc attccgcaga aaggcgtggt
      841 ggataaacgt ggcaaacgta aaaaccgtcc ggcgcgtaaa gcgagcctga gcctgcgtag
      901 cggccgtatt accctgaaac agggcaacat taccctgaac gcggtgctgg ccgaagaaat
      961 taatccgccg aaaggcgaaa ccccgctgaa atggctgctg ctgaccagcg agccggtgga
     1021 aagtctggcc caagcgctgc gtgtgattga tatttatacc catcgttggc gcattgaaga
     1081 atttcacaaa gcgtggaaaa cgggtgcggg tgcggaacgt cagcgtatgg aagaaccgga
     1141 taacctggaa cgtatggtga gcattctgag ctttgtggcg gtgcgtctgc tgcaactgcg
     1201 tgaatctttt actccgccgc aagcactgcg tgcgcagggc ctgctgaaag aagcggaaca
     1261 cgttgaaagc cagagcgcgg aaaccgtgct gaccccggat gaatgccaac tgctgggcta
     1321 tctggataaa ggcaaacgca aacgcaaaga aaaagcgggc agcctgcaat gggcgtatat
     1381 ggcgattgcg cgtctgggcg gctttatgga tagcaaacgt accggcattg cgagctgggg
     1441 tgcgctgtgg gaaggttggg aagcgctgca aagcaaactg gatggctttc tggccgcgaa
     1501 agacctgatg gcgcagggca ttaaaatctg acagtaaagg tggatacgga tccgaattcg
     1561 agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
     1621 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1681 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1741 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1801 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1861 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1921 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1981 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2041 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     2101 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     2161 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     2221 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2281 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     2341 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     2401 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     2461 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     2521 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     2581 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     2641 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     2701 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2761 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     2821 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     2881 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     2941 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     3001 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     3061 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     3121 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     3181 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3241 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3301 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3361 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3421 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3481 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3541 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3601 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3661 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3721 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3781 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3841 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3901 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3961 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4021 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     4081 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     4141 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4201 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4261 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     4321 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     4381 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     4441 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     4501 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     4561 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     4621 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4681 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4741 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4801 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4861 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4921 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     4981 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     5041 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     5101 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     5161 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     5221 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5281 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5341 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     5401 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     5461 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     5521 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     5581 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     5641 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5701 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5761 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5821 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     5881 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     5941 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     6001 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     6061 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     6121 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     6181 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     6241 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     6301 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     6361 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     6421 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     6481 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     6541 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     6601 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     6661 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     6721 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     6781 aaa
//
