LOCUS       sequence-217501-        4909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression vector for untagged E. coli Cas1 + Cas2 with 
            CRISPR array, leader ggtTg mutant.
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: pAW216
FEATURES             Location/Qualifiers
     source          1..4909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..53
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        34..52
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    53..77
                     /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             1363..1407
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1445..1463)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1459..1506
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1677..2468)
                     /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(2469..2560)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2608..3346)
                     /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(3737..4819)
                     /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     4781..4800
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4820..4897)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gctctccctt atgcgactcc tgcattagga aattaatacg actcactata ggggaattgt
       61 gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag gagatatacc
      121 atggcgatga cctggcttcc ccttaatccc attccactca aagatcgcgt ctccatgatc
      181 tttctgcaat atgggcagat cgatgtaata gatggcgcgt ttgtacttat cgacaagaca
      241 gggatccgca ctcatattcc tgttggctcg gttgcctgca tcatgctgga acctggtaca
      301 cgggtttcgc atgcagctgt acgcctggct gcgcaagttg gaacattgtt ggtatgggtg
      361 ggggaagcgg gcgttcgtgt ttatgcttct ggtcagcctg gaggtgcgcg ttcagataag
      421 ctgctctatc aggcaaaact tgctctggat gaagatttgc gtctgaaggt cgtacgtaaa
      481 atgtttgaac ttcggtttgg agaacctgcg cctgcccggc gctccgtaga gcaactcaga
      541 ggtatagaag gcagtcgcgt gcgggcaacc tacgcacttc tggcgaagca atacggcgtg
      601 acatggaatg gacgtcgcta cgatccgaaa gactgggaaa agggcgatac gatcaaccaa
      661 tgcattagcg ctgcaacttc ctgtttatac ggcgtaactg aagcggcgat acttgcagct
      721 ggttatgcac cagctattgg gtttgtgcat acaggaaagc ctctttcctt tgtttacgat
      781 attgcagaca tcattaaatt tgacactgtt gtaccgaaag cttttgagat agcgcgtcgt
      841 aaccctggtg agccggaccg ggaagtccgt ttggcgtgca gggatatttt tcgcagtagt
      901 aaaacattag ccaaattgat tccgcttata gaggacgtgc ttgccgctgg agaaatacaa
      961 ccgccggccc cacctgaaga tgcacagcct gttgccattc cgcttcctgt ttcactggga
     1021 gatgcaggcc atcgctcgag ctgaaggaga tataccatga gtatgttggt cgtggtcact
     1081 gaaaatgtac ctccgcgctt acgaggcaga ttagccatct ggttgttgga ggtacgtgca
     1141 ggggtatatg taggtgatgt atccgcaaaa attcgtgaaa tgatctggga acaaatagct
     1201 ggactggcgg aagaaggcaa tgtagtgatg gcatgggcaa cgaatacgga aacgggattt
     1261 gagttccaga catttgggtt aaacaggcgt accccggtag atttggatgg tttaaggttg
     1321 gtgtcttttt tacctgtttg agcggccgca ctcgagtctg gtaaagaaac cgctgctgcg
     1381 aaatttgaac gccagcacat ggactcgtct actagcgcag cttaattaac ctaggctgct
     1441 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     1501 tttttgctga aacctcaggc atttgagaag cacacggtca cactgcttcc ggtagtcaat
     1561 aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc tgaaccgacg
     1621 accgggtcat cgtggccgga tcttgcggcc cctcggcttg aacgaattgt tagacattat
     1681 ttgccgacta ccttggtgat ctcgcctttc acgtagtgga caaattcttc caactgatct
     1741 gcgcgcgagg ccaagcgatc ttcttcttgt ccaagataag cctgtctagc ttcaagtatg
     1801 acgggctgat actgggccgg caggcgctcc attgcccagt cggcagcgac atccttcggc
     1861 gcgattttgc cggttactgc gctgtaccaa atgcgggaca acgtaagcac tacatttcgc
     1921 tcatcgccag cccagtcggg cggcgagttc catagcgtta aggtttcatt tagcgcctca
     1981 aatagatcct gttcaggaac cggatcaaag agttcctccg ccgctggacc taccaaggca
     2041 acgctatgtt ctcttgcttt tgtcagcaag atagccagat caatgtcgat cgtggctggc
     2101 tcgaagatac ctgcaagaat gtcattgcgc tgccattctc caaattgcag ttcgcgctta
     2161 gctggataac gccacggaat gatgtcgtcg tgcacaacaa tggtgacttc tacagcgcgg
     2221 agaatctcgc tctctccagg ggaagccgaa gtttccaaaa ggtcgttgat caaagctcgc
     2281 cgcgttgttt catcaagcct tacggtcacc gtaaccagca aatcaatatc actgtgtggc
     2341 ttcaggccgc catccactgc ggagccgtac aaatgtacgg ccagcaacgt cggttcgaga
     2401 tggcgctcga tgacgccaac tacctctgat agttgagtcg atacttcggc gatcaccgct
     2461 tccctcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     2521 agcggataca tatttgaatg tatttagaaa aataaacaaa tagctagctc actcggtcgc
     2581 tacgctccgg gcgtgagact gcggcgggcg ctgcggacac atacaaagtt acccacagat
     2641 tccgtggata agcaggggac taacatgtga ggcaaaacag cagggccgcg ccggtggcgt
     2701 ttttccatag gctccgccct cctgccagag ttcacataaa cagacgcttt tccggtgcat
     2761 ctgtgggagc cgtgaggctc aaccatgaat ctgacagtac gggcgaaacc cgacaggact
     2821 taaagatccc caccgtttcc ggcgggtcgc tccctcttgc gctctcctgt tccgaccctg
     2881 ccgtttaccg gatacctgtt ccgcctttct cccttacggg aagtgtggcg ctttctcata
     2941 gctcacacac tggtatctcg gctcggtgta ggtcgttcgc tccaagctgg gctgtaagca
     3001 agaactcccc gttcagcccg actgctgcgc cttatccggt aactgttcac ttgagtccaa
     3061 cccggaaaag cacggtaaaa cgccactggc agcagccatt ggtaactggg agttcgcaga
     3121 ggatttgttt agctaaacac gcggttgctc ttgaagtgtg cgccaaagtc cggctacact
     3181 ggaaggacag atttggttgc tgtgctctgc gaaagccagt taccacggtt aagcagttcc
     3241 ccaactgact taaccttcga tcaaaccacc tccccaggtg gttttttcgt ttacagggca
     3301 aaagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctactgaacc
     3361 gctctagata cctttttaat caatggatta agtactcttt aacataatgg atgtgttgtt
     3421 tgtgtgatac tataaagttg gttgattgtg actggcttaa aaaatcatta attaataata
     3481 ggttatgttt agagtgttcc ccgcgccagc ggggataaac cgagcacaaa tatcatcgct
     3541 caatctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     3601 gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg
     3661 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     3721 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3781 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     3841 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     3901 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     3961 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     4021 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     4081 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     4141 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     4201 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     4261 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     4321 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     4381 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     4441 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     4501 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     4561 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     4621 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     4681 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     4741 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     4801 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     4861 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgac
//
