LOCUS       sequence-345504-        7557 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His14-MBP-TEV-AloTnpB-2 with 3' extension 
            derived from locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7557)
  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 7557)
  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: pHS0766 pET45b(+) His14-MBP-TEV-AloTnpB-2 3'
            e...
FEATURES             Location/Qualifiers
     source          1..7557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      768..815
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      852..1307
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(939..958)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1149..1170
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1422..2213,2214..2282))
                     /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     2045..2064
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2283..2375)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2456..3044
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2945..2964
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3198..3215
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3230..3369
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3295..3314)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3455..3477
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3471..3662)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4234..5316)
                     /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     5278..5297
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5317..5394)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5600..5619
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5707..5726
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5707..5725
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5726..5750
                     /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             5765..5787
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5872..6969
                     /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      6826..6835
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     6943..6966
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             7003..7023
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
ORIGIN
        1 accgtcgaga tcgccgaggc caccccggtc gaaccggctg gtttcctcgg tgtggatttg
       61 ggcatcgtgc agatcgccac cgactccgac ggcaccgtct acgcgggcga acagctgaac
      121 cggtaccggc gccgccagat ccggttgcgc gccaagttgc aggccaagaa gaccgaatcc
      181 gcccgccggc tgctggtcaa acgcgcccgc cgggagtcac ggcacgctac gaacgtcaac
      241 catgtgatct cgaaaagcat tgtggccgag gctgaacgca cctcgcgtgg tatcgccgtc
      301 gaagacctga cggggatccg cgccagggtc cggctccgca agccccaacg ggccgcactg
      361 cattcttgga gcttcgccca actcggcggc ttcttgacct acaaggcccg ccgggccggt
      421 atccctctgg tccaggtcga tccccggtac acgtcgcaga cgtgctcggc atgcggtcac
      481 cgggacaaga ggaaccggcc cgaccaggcc acgttcatct gccggtcgtg tggagtcgtt
      541 gcccacgcgg acgtcaatgc tgccgtcaac atcgccgcgc gtggtgtcga cgtctggggc
      601 gcagtcagcc gcccgtacgt ggcctgacct cgcagccagc gggagcagag atccacaagc
      661 tcgcccattc atgggcgggt agttgactgc gagacgctgc aaccggtgct cacccaggta
      721 ctcggcacgc tggaccagcc gcacaccgtc gacacgatgg ccgagttcta gcataacccc
      781 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
      841 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
      901 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      961 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1021 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1081 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1141 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1201 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1261 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttctg gcggcacgat
     1321 ggcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     1381 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     1441 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1501 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1561 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1621 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1681 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1741 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1801 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1861 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1921 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1981 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     2041 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     2101 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     2161 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     2221 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     2281 atactcttcc tttttcaatc atgattgaag catttatcag ggttattgtc tcatgagcgg
     2341 atacatattt gaatgtattt agaaaaataa acaaataggt catgaccaaa atcccttaac
     2401 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2461 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2521 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2581 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2641 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2701 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2761 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2821 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2881 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2941 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3001 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3061 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3121 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3181 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3241 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     3301 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3361 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3421 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3481 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3541 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3601 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3661 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3721 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     3781 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3841 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3901 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3961 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     4021 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4081 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac
     4141 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     4201 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4261 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4321 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     4381 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     4441 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     4501 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     4561 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     4621 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     4681 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     4741 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     4801 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     4861 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4921 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4981 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     5041 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     5101 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     5161 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     5221 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     5281 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     5341 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     5401 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     5461 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     5521 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     5581 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     5641 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg
     5701 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
     5761 taattttgtt taactttaag aaggagatat accatgagca agcatcacca tcattcaggc
     5821 catcaccata ccggacacca ccatcattca ggcagtcatc accataccgg gaaaactgaa
     5881 gaaggtaaac tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc
     5941 ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg
     6001 gaagagaaat tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca
     6061 cacgaccgct ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa
     6121 gcgttccagg acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg
     6181 attgcttacc cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg
     6241 aacccgccaa aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt
     6301 aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct
     6361 gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg
     6421 gataacgctg gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac
     6481 atgaatgcag acactgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg
     6541 atgaccatca acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt
     6601 gtaacggtac tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc
     6661 gcaggtatta acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat
     6721 ctgctgactg atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg
     6781 ctgaagtctt acgaggaaga gttggcgaaa gatccacgta ttgccgccac catggaaaac
     6841 gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg
     6901 cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac
     6961 gcgcagacta attcgagctc gaacaacaac aacggtggtg gagagaacct gtattttcag
     7021 ggtggaggta gtatgaagct ggtggtgcag gtgaagcttc agccgacggc cgaacaggcg
     7081 tcgatgttgg aggcgaccct gcgtgcttgc aacacggccg cgaacgaggt tgctcaggtg
     7141 gcccgccggg cgcgggtcta ccgcaactac gacctgcgca agcatgtgta cgcgggcatc
     7201 aaagccgatc atcggctcgg gtcccaggcc gcccagcatg tgatcaagaa ggtttgtgac
     7261 gcgtacaaga ccctcacgtc gaatctgcgg gccggcaact acggcccgcc ggatgcgaag
     7321 cggtaccggc gggtgtccac cgagccggtc cggttccggt ggcaggccgc ccagccgtac
     7381 gacgcgcgga tgctgtcgtg gcagcacgac gcccgtacgg tgtcgatctg gaccgtggcc
     7441 ggccggatga agaacatcgc ctacaccggc tctcccgacc agctcaaagc cgtcgccgag
     7501 ttgccggtcg gtgaatgcga tctggtgcac cgcgacggca tgtggttgct ctacgcg
//
