LOCUS       sequence-218411-        7554 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for His-MBP-E. coli Cas1 R138A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7554)
  AUTHORS   Wright AV, Liu JJ, Knott GJ, Doxzen KW, Nogales E, Doudna JA
  TITLE     Structures of the CRISPR genome integration complex.
  JOURNAL   Science. 2017 Sep 15;357(6356):1113-1118. doi: 
            10.1126/science.aao0679. Epub 2017 Jul 20.
  PUBMED    28729350
REFERENCE   2  (bases 1 to 7554)
  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.aao0679"; journalName: "Science"; date: 
            "2017-09-15- 15"; volume: "357"; issue: "6356"; pages: "1113-1118"
COMMENT     Sequence Label: pAW181
FEATURES             Location/Qualifiers
     source          1..7554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     564..583
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(603..680)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     886..905
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     995..1014
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        995..1013
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1014..1038
                     /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             1053..1075
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1094..1111
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1118..2215
                     /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      2072..2081
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2189..2212
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    2234..2258
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2279..2299
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(3237..3261)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3275..3292
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3345..3363)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     rep_origin      3442..3897
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3529..3548)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3739..3760
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3923..4027
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4028..4096,4097..4888)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4246..4265)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5059..5647
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5548..5567
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5801..5818
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5833..5972
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5898..5917)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6058..6080
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6074..6265)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7074..7554,1..602))
                     /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 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
       61 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
      121 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
      181 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
      241 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
      301 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
      361 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
      421 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
      481 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
      541 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
      601 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
      661 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
      721 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
      781 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
      841 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      901 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      961 gaggatcgag atccagatct cgatcccgcg aaattaatac gactcactat aggggaattg
     1021 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     1081 catgggcagc agccatcatc atcatcatca cggtaccaaa actgaagaag gtaaactggt
     1141 aatctggatt aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga
     1201 gaaagatacc ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc
     1261 acaggttgcg gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg
     1321 tggctacgct caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa
     1381 gctgtatccg tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat
     1441 cgctgttgaa gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac
     1501 ctgggaagag atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat
     1561 gttcaacctg caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc
     1621 gttcaagtat gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc
     1681 gaaagcgggt ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac
     1741 cgattactcc atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg
     1801 cccgtgggca tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc
     1861 gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc
     1921 cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga
     1981 aggtctggaa gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga
     2041 ggaagagttg gcgaaagatc cacgtattgc cgccaccatg gaaaacgccc agaaaggtga
     2101 aatcatgccg aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat
     2161 caacgccgcc agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactggtac
     2221 cggatcttac atcacaagtt tgtacaaaaa agcaggctcc gcggccgccg tactcgagga
     2281 aaacctgtat tttcagggct ccatgacctg gcttcccctt aatcccattc cactcaaaga
     2341 tcgcgtctcc atgatctttc tgcaatatgg gcagatcgat gtaatagatg gcgcgtttgt
     2401 acttatcgac aagacaggga tccgcactca tattcctgtt ggctcggttg cctgcatcat
     2461 gctggaacct ggtacacggg tttcgcatgc agctgtacgc ctggctgcgc aagttggaac
     2521 attgttggta tgggtggggg aagcgggcgt tcgtgtttat gcttctggtc agcctggagg
     2581 tgcgcgttca gataagctgc tctatcaggc aaaacttgct ctggatgaag atttgcgtct
     2641 gaaggtcgta cgtaaaatgt ttgaacttcg gtttggagaa cctgcgcctg cccggcgctc
     2701 cgtagagcaa ctcgcaggta tagaaggcag tcgcgtgcgg gcaacctacg cacttctggc
     2761 gaagcaatac ggcgtgacat ggaatggacg tcgctacgat ccgaaagact gggaaaaggg
     2821 cgatacgatc aaccaatgca ttagcgctgc aacttcctgt ttatacggcg taactgaagc
     2881 ggcgatactt gcagctggtt atgcaccagc tattgggttt gtgcatacag gaaagcctct
     2941 ttcctttgtt tacgatattg cagacatcat taaatttgac actgttgtac cgaaagcttt
     3001 tgagatagcg cgtcgtaacc ctggtgagcc ggaccgggaa gtccgtttgg cgtgcaggga
     3061 tatttttcgc agtagtaaaa cattagccaa attgattccg cttatagagg acgtgcttgc
     3121 cgctggagaa atacaaccgc cggccccacc tgaagatgca cagcctgttg ccattccgct
     3181 tcctgtttca ctgggagatg caggccatcg gagtagctga aagggtgggc gcgccgaccc
     3241 agctttcttg tacaaagtgg tgatgtacct cgagcaccac caccaccacc actgagatcc
     3301 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     3361 agcataaccc cttggggcct ctaaacgggt cttgaggggt ttttgctgaa aggaggaact
     3421 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3481 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3541 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3601 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3661 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3721 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3781 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3841 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     3901 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3961 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4021 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4081 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     4141 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     4201 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4261 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4321 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4381 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4441 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4501 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4561 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4621 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4681 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4741 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4801 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4861 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4921 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     4981 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5041 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5101 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5161 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     5221 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5281 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5341 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5401 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5461 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5521 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5581 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5641 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5701 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     5761 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     5821 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     5881 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     5941 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     6001 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     6061 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     6121 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     6181 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     6241 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     6301 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     6361 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     6421 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     6481 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     6541 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     6601 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     6661 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     6721 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     6781 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     6841 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     6901 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     6961 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     7021 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     7081 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     7141 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     7201 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     7261 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     7321 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     7381 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     7441 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     7501 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgc
//
