LOCUS       sequence-238613-        7554 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas1 cloned in pHMGWA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7554)
  AUTHORS   Nunez JK, Kranzusch PJ, Noeske J, Wright AV, Davies CW, Doudna JA
  TITLE     Cas1-Cas2 complex formation mediates spacer acquisition during 
            CRISPR-Cas adaptive immunity.
  JOURNAL   Nat Struct Mol Biol. 2014 Jun;21(6):528-34. doi: 10.1038/nsmb.2820. 
            Epub 2014 May 4.
  PUBMED    24793649
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: "10.1038/nsmb.2820";
            journalName: "Nat Struct Mol Biol"; date: "2014-06"; volume: "21"; 
            issue: "6"; pages: "528-34"
COMMENT     Sequence Label: pHMGWA-Cas1
FEATURES             Location/Qualifiers
     source          1..7554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     550..569
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(589..666)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     872..891
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     981..1000
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        981..999
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1000..1024
                     /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             1039..1061
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1080..1097
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1104..2201
                     /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      2058..2067
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     2175..2198
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    2220..2244
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2265..2285
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(3223..3247)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3261..3278
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3331..3349)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     rep_origin      3428..3883
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3515..3534)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3725..3746
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3909..4013
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4014..4082,4083..4874)
                     /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(4232..4251)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5045..5633
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5534..5553
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5787..5804
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5819..5958
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5884..5903)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6044..6066
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6060..6251)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7060..7554,1..588))
                     /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 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
       61 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      121 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      181 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      241 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      301 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      361 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      421 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      481 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      541 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
      601 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
      661 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
      721 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
      781 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
      841 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
      901 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcc
      961 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     1021 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc
     1081 atcatcatca tcatcacggt accaaaactg aagaaggtaa actggtaatc tggattaacg
     1141 gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa gataccggaa
     1201 ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag gttgcggcaa
     1261 ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc tacgctcaat
     1321 ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg tatccgttta
     1381 cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct gttgaagcgt
     1441 tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc
     1501 cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag
     1561 aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa
     1621 acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga
     1681 ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat tactccatcg
     1741 cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg tgggcatggt
     1801 ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc ttcaagggtc
     1861 aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca
     1921 aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg
     1981 ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga
     2041 aagatccacg tattgccgcc accatggaaa acgcccagaa aggtgaaatc atgccgaaca
     2101 tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg
     2161 gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac tggtaccgga tcttacatca
     2221 caagtttgta caaaaaagca ggctccgcgg ccgccgtact cgaggaaaac ctgtattttc
     2281 agggctccat gacctggctt ccccttaatc ccattccact caaagatcgc gtctccatga
     2341 tctttctgca atatgggcag atcgatgtaa tagatggcgc gtttgtactt atcgacaaga
     2401 cagggatccg cactcatatt cctgttggct cggttgcctg catcatgctg gaacctggta
     2461 cacgggtttc gcatgcagct gtacgcctgg ctgcgcaagt tggaacattg ttggtatggg
     2521 tgggggaagc gggcgttcgt gtttatgctt ctggtcagcc tggaggtgcg cgttcagata
     2581 agctgctcta tcaggcaaaa cttgctctgg atgaagattt gcgtctgaag gtcgtacgta
     2641 aaatgtttga acttcggttt ggagaacctg cgcctgcccg gcgctccgta gagcaactca
     2701 gaggtataga aggcagtcgc gtgcgggcaa cctacgcact tctggcgaag caatacggcg
     2761 tgacatggaa tggacgtcgc tacgatccga aagactggga aaagggcgat acgatcaacc
     2821 aatgcattag cgctgcaact tcctgtttat acggcgtaac tgaagcggcg atacttgcag
     2881 ctggttatgc accagctatt gggtttgtgc atacaggaaa gcctctttcc tttgtttacg
     2941 atattgcaga catcattaaa tttgacactg ttgtaccgaa agcttttgag atagcgcgtc
     3001 gtaaccctgg tgagccggac cgggaagtcc gtttggcgtg cagggatatt tttcgcagta
     3061 gtaaaacatt agccaaattg attccgctta tagaggacgt gcttgccgct ggagaaatac
     3121 aaccgccggc cccacctgaa gatgcacagc ctgttgccat tccgcttcct gtttcactgg
     3181 gagatgcagg ccatcggagt agctgaaagg gtgggcgcgc cgacccagct ttcttgtaca
     3241 aagtggtgat gtacctcgag caccaccacc accaccactg agatccggct gctaacaaag
     3301 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     3361 gggcctctaa acgggtcttg aggggttttt gctgaaagga ggaactatat ccggattggc
     3421 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     3481 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     3541 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     3601 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3661 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3721 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3781 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3841 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     3901 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     3961 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4021 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4081 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4141 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4201 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4261 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4321 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4381 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4441 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4501 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     4561 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4621 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4681 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4741 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4801 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4861 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4921 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     4981 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5041 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5101 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5161 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     5221 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5281 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5341 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5401 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5461 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5521 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5581 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5641 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5701 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5761 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5821 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     5881 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     5941 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     6001 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     6061 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     6121 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     6181 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     6241 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     6301 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     6361 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     6421 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     6481 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     6541 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     6601 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     6661 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     6721 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     6781 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     6841 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     6901 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     6961 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     7021 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     7081 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     7141 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     7201 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     7261 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     7321 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     7381 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     7441 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     7501 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacg
//
