LOCUS       sequence-345507-        7387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His14-MBP-TEV-AmaTnpB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7387)
  AUTHORS   Altae-Tran H, Kannan S, Demircioglu FE, Oshiro R, Nety SP, McKay LJ,
            Dlakic M, Inskeep WP, Makarova KS, Macrae RK, Koonin EV, Zhang F
  TITLE     The widespread IS200/IS605 transposon family encodes diverse 
            programmable RNA-guided endonucleases.
  JOURNAL   Science. 2021 Oct;374(6563):57-65. doi: 10.1126/science.abj6856. 
            Epub 2021 Sep 9.
  PUBMED    34591643
REFERENCE   2  (bases 1 to 7387)
  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"; doi: 
            "10.1126/science.abj6856"; journalName: "Science"; date: "2021-10"; 
            volume: "374"; issue: "6563"; pages: "57-65"
COMMENT     Sequence Label: pHS0923 pET45b(+) His14-MBP-TEV-AmaTnpB
FEATURES             Location/Qualifiers
     source          1..7387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     969..988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1008..1085)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1291..1310
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1398..1417
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1398..1416
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1417..1441
                     /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)."
     RBS             1456..1478
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1563..2660
                     /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="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2517..2526
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2634..2657
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2694..2714
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      3846..3893
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3930..4385
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4017..4036)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4227..4248
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4500..5291,5292..5360))
                     /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     5123..5142
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5361..5453)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5534..6122
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6023..6042
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6276..6293
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6308..6447
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6373..6392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6533..6555
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6549..6740)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7312..7387,1..1007))
                     /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"
ORIGIN
        1 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
       61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
      181 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
      241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     1081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     1141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     1201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     1261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     1321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga
     1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1441 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgagc aagcatcacc
     1501 atcattcagg ccatcaccat accggacacc accatcattc aggcagtcat caccataccg
     1561 ggaaaactga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc
     1621 tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc
     1681 cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta
     1741 tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca
     1801 ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca
     1861 acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag
     1921 atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga
     1981 aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc
     2041 tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag
     2101 acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta
     2161 aaaacaaaca catgaatgca gacactgatt actccatcgc agaagctgcc tttaataaag
     2221 gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag
     2281 tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg
     2341 gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc
     2401 tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg
     2461 gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca
     2521 ccatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct
     2581 ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag
     2641 ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacggtggt ggagagaacc
     2701 tgtattttca gggtggaggt agtatgggga ggaaacgcgc tcttattgta ttgaacgatt
     2761 tgtgctataa taacggcatg aacctgacgt tgatggtcaa gctgctaccc acaactgaac
     2821 agcaccaagc gttgcttgag acgatggaac ggttcaatgc ggcttgcaat gcgattgcgg
     2881 aagtggcttt cgaacatcgc actgcaaaca agatccggtt gcaaaagctc gtgtacgaca
     2941 gtatccgcaa ggaatttggg ctttcagccc aaatgaccgt ccgtgctatc gctaaggcgt
     3001 gcgaagcgta caagcgagac aagtcgataa agccgacatt caagccgcat ggcgccatcg
     3061 tatatgacca acgcctcctt tcttggaaag gtcttgatcg cgtgtccatc ctaacgcttg
     3121 gcggccgcat cttggtcccc attttgtttg gcgagtacca ggctgcacgc ctccagcgga
     3181 ttcgtggaca agccgacctg atttaccgcg atggcacctt ctacctcgct gtcgttgtgg
     3241 atgtcccaga tccaccacaa ggtgctccaa atggatttct tggcgtggac ttgggcatta
     3301 agaacatcgc gacagacagc gacggcgagg tgttttctgg agggcacgtg aacggactgc
     3361 gccatcgcca cgcacgcctt cgccaacgcc tgcaatcgaa aggcacaaag tctgccaagc
     3421 gtctgctgaa gaagcgtcgc cgcaaggaag cccgtttcgc caccaacgta aaccatcgca
     3481 ttgcgaaagt gctggttgcg aaagccaaag acaccgggcg cgggattgca cttgaagatc
     3541 tcaagggcat tcgtgaccgg ataacggttc gcaaggctca gcgtcgcacc cagcattcct
     3601 gggcattcca tcagcttcgc ttcttcatcg agtacaaggc acgtttggcg ggtgtgcccg
     3661 tggtgttcgt cgatccgcgc aatacatctc gaacatgtcc aagctgcggt catgcggaca
     3721 aacgcaatcg ccccacaagg gataattttg agtgtgtcga atgtgggttc gctggcccag
     3781 ccgacaccat cgctgcggtg aacattcgcc gtagggctga agtcatgcag ccgtacgcgg
     3841 tatagctagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     3901 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     3961 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     4021 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4081 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4141 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4201 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4261 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4321 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     4381 aatttctggc ggcacgatgg catgagatta tcaaaaagga tcttcaccta gatcctttta
     4441 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4501 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4561 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4621 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4681 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4741 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4801 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4861 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4921 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4981 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5041 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5101 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5161 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5221 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5281 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5341 cggaaatgtt gaatactcat actcttcctt tttcaatcat gattgaagca tttatcaggg
     5401 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggtca
     5461 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5521 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5581 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5641 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     5701 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5761 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5821 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5881 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5941 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6001 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6061 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6121 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6181 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6241 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6301 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     6361 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     6421 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     6481 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     6541 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     6601 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     6661 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     6721 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     6781 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     6841 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     6901 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     6961 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     7021 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     7081 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     7141 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcta
     7201 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     7261 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     7321 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     7381 agaggcg
//
