LOCUS       sequence-213838-        4909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for untagged E. coli Cas1 + Cas2 with 
            CRISPR array, leader IHF binding site flip.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4909)
  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 4909)
  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: pAW218
FEATURES             Location/Qualifiers
     source          1..4909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             1313..1357
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1395..1413)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1409..1456
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1627..2418)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2419..2510)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2558..3296)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(3687..4769)
                     /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     4731..4750
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4770..4847)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4893..4909,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4893..4909,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atggcgatga cctggcttcc ccttaatccc attccactca aagatcgcgt
      121 ctccatgatc tttctgcaat atgggcagat cgatgtaata gatggcgcgt ttgtacttat
      181 cgacaagaca gggatccgca ctcatattcc tgttggctcg gttgcctgca tcatgctgga
      241 acctggtaca cgggtttcgc atgcagctgt acgcctggct gcgcaagttg gaacattgtt
      301 ggtatgggtg ggggaagcgg gcgttcgtgt ttatgcttct ggtcagcctg gaggtgcgcg
      361 ttcagataag ctgctctatc aggcaaaact tgctctggat gaagatttgc gtctgaaggt
      421 cgtacgtaaa atgtttgaac ttcggtttgg agaacctgcg cctgcccggc gctccgtaga
      481 gcaactcaga ggtatagaag gcagtcgcgt gcgggcaacc tacgcacttc tggcgaagca
      541 atacggcgtg acatggaatg gacgtcgcta cgatccgaaa gactgggaaa agggcgatac
      601 gatcaaccaa tgcattagcg ctgcaacttc ctgtttatac ggcgtaactg aagcggcgat
      661 acttgcagct ggttatgcac cagctattgg gtttgtgcat acaggaaagc ctctttcctt
      721 tgtttacgat attgcagaca tcattaaatt tgacactgtt gtaccgaaag cttttgagat
      781 agcgcgtcgt aaccctggtg agccggaccg ggaagtccgt ttggcgtgca gggatatttt
      841 tcgcagtagt aaaacattag ccaaattgat tccgcttata gaggacgtgc ttgccgctgg
      901 agaaatacaa ccgccggccc cacctgaaga tgcacagcct gttgccattc cgcttcctgt
      961 ttcactggga gatgcaggcc atcgctcgag ctgaaggaga tataccatga gtatgttggt
     1021 cgtggtcact gaaaatgtac ctccgcgctt acgaggcaga ttagccatct ggttgttgga
     1081 ggtacgtgca ggggtatatg taggtgatgt atccgcaaaa attcgtgaaa tgatctggga
     1141 acaaatagct ggactggcgg aagaaggcaa tgtagtgatg gcatgggcaa cgaatacgga
     1201 aacgggattt gagttccaga catttgggtt aaacaggcgt accccggtag atttggatgg
     1261 tttaaggttg gtgtcttttt tacctgtttg agcggccgca ctcgagtctg gtaaagaaac
     1321 cgctgctgcg aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac
     1381 ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1441 cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca cactgcttcc
     1501 ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc
     1561 tgaaccgacg accgggtcat cgtggccgga tcttgcggcc cctcggcttg aacgaattgt
     1621 tagacattat ttgccgacta ccttggtgat ctcgcctttc acgtagtgga caaattcttc
     1681 caactgatct gcgcgcgagg ccaagcgatc ttcttcttgt ccaagataag cctgtctagc
     1741 ttcaagtatg acgggctgat actgggccgg caggcgctcc attgcccagt cggcagcgac
     1801 atccttcggc gcgattttgc cggttactgc gctgtaccaa atgcgggaca acgtaagcac
     1861 tacatttcgc tcatcgccag cccagtcggg cggcgagttc catagcgtta aggtttcatt
     1921 tagcgcctca aatagatcct gttcaggaac cggatcaaag agttcctccg ccgctggacc
     1981 taccaaggca acgctatgtt ctcttgcttt tgtcagcaag atagccagat caatgtcgat
     2041 cgtggctggc tcgaagatac ctgcaagaat gtcattgcgc tgccattctc caaattgcag
     2101 ttcgcgctta gctggataac gccacggaat gatgtcgtcg tgcacaacaa tggtgacttc
     2161 tacagcgcgg agaatctcgc tctctccagg ggaagccgaa gtttccaaaa ggtcgttgat
     2221 caaagctcgc cgcgttgttt catcaagcct tacggtcacc gtaaccagca aatcaatatc
     2281 actgtgtggc ttcaggccgc catccactgc ggagccgtac aaatgtacgg ccagcaacgt
     2341 cggttcgaga tggcgctcga tgacgccaac tacctctgat agttgagtcg atacttcggc
     2401 gatcaccgct tccctcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     2461 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa tagctagctc
     2521 actcggtcgc tacgctccgg gcgtgagact gcggcgggcg ctgcggacac atacaaagtt
     2581 acccacagat tccgtggata agcaggggac taacatgtga ggcaaaacag cagggccgcg
     2641 ccggtggcgt ttttccatag gctccgccct cctgccagag ttcacataaa cagacgcttt
     2701 tccggtgcat ctgtgggagc cgtgaggctc aaccatgaat ctgacagtac gggcgaaacc
     2761 cgacaggact taaagatccc caccgtttcc ggcgggtcgc tccctcttgc gctctcctgt
     2821 tccgaccctg ccgtttaccg gatacctgtt ccgcctttct cccttacggg aagtgtggcg
     2881 ctttctcata gctcacacac tggtatctcg gctcggtgta ggtcgttcgc tccaagctgg
     2941 gctgtaagca agaactcccc gttcagcccg actgctgcgc cttatccggt aactgttcac
     3001 ttgagtccaa cccggaaaag cacggtaaaa cgccactggc agcagccatt ggtaactggg
     3061 agttcgcaga ggatttgttt agctaaacac gcggttgctc ttgaagtgtg cgccaaagtc
     3121 cggctacact ggaaggacag atttggttgc tgtgctctgc gaaagccagt taccacggtt
     3181 aagcagttcc ccaactgact taaccttcga tcaaaccacc tccccaggtg gttttttcgt
     3241 ttacagggca aaagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3301 ctactgaacc gctctagata cctttttaat caatggatta agtactcttt aacataatgg
     3361 atgtgttgtt tgtgtgatac tataaagttg gtagattgtg actggcttca taacctatta
     3421 ttaattaatg attttttttt agagtgttcc ccgcgccagc ggggataaac cgagcacaaa
     3481 tatcatcgct caatctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     3541 agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg
     3601 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     3661 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3721 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     3781 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     3841 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     3901 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     3961 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4021 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4081 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4141 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4201 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4261 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     4321 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     4381 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     4441 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     4501 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     4561 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     4621 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     4681 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     4741 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     4801 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     4861 ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata
//
