LOCUS       sequence-150476-        7356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a set of wild-type Cas1+2 and a mutant set of Cas1+2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7356)
  AUTHORS   Shipman SL, Nivala J, Macklis JD, Church GM
  TITLE     Molecular recordings by directed CRISPR spacer acquisition.
  JOURNAL   Science. 2016 Jun 9. pii: aaf1175.
  PUBMED    27284167
REFERENCE   2  (bases 1 to 7356)
  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"; journalName: "Science. 
            2016 Jun 9. pii: aaf1175."
COMMENT     Sequence Label: pWUR 1+2 tetO mut89
FEATURES             Location/Qualifiers
     source          1..7356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     primer_bind     complement(1332..1349)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1502..1588
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(1750..1768,1769..1774,1775..1791,1792..1797,
                     1798..1823)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1750..1768
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1775..1793
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             3298..3306
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             3314..3937
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(3387..3406)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             3958..4002
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4040..4058)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4054..4101
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4272..5063)
                     /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(5064..5155)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5203..5941)
                     /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(6151..7233)
                     /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     7195..7214
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7234..7311)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctgta gaaataattt
       61 tgtttaactt taataaggag atataccatg acctggcttc cccttaatcc cattccactc
      121 aaagatcgcg tctccatgat ctttctgcaa tatgggcaga tcgatgtaat agatggcgcg
      181 tttgtactta tcgacaagac agggatccgc actcatattc ctgttggctc ggttgcctgc
      241 atcatgctgg aacctggtac acgggtttcg catgcagctg tacgcctggc tgcgcaagtt
      301 ggaacattgt tggtatgggt gggggaagcg ggcgttcgtg tttatgcttc tggtcagcct
      361 ggaggtgcgc gttcagataa gctgctctat caggcaaaac ttgctctgga tgaagatttg
      421 cgtctgaagg tcgtacgtaa aatgtttgaa cttcggtttg gagaacctgc gcctgcccgg
      481 cgctccgtag agcaactcag aggtatagaa ggcagtcgcg tgcgggcaac ctacgcactt
      541 ctggcgaagc aatacggcgt gacatggaat ggacgtcgct acgatccgaa agactgggaa
      601 aagggcgata cgatcaacca atgcattagc gctgcaactt cctgtttata cggcgtaact
      661 gaagcggcga tacttgcagc tggttatgca ccagctattg ggtttgtgca tacaggaaag
      721 cctctttcct ttgtttacga tattgcagac atcattaaat ttgacactgt tgtaccgaaa
      781 gcttttgaga tagcgcgtcg taaccctggt gagccggacc gggaagtccg tttggcgtgc
      841 agggatattt ttcgcagtag taaaacatta gccaaattga ttccgcttat agaggacgtg
      901 cttgccgctg gagaaataca accgccggcc ccacctgaag atgcacagcc tgttgccatt
      961 ccgcttcctg tttcactggg agatgcaggc catcggagta gctgaaatga 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 agcggccgcg gctgttttgg cggatgagag
     1321 aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat
     1381 ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa
     1441 cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca
     1501 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     1561 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca
     1621 acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca
     1681 gaaggccatc cgtcaggatg gcctttttgc gtttctacaa actctgctag caagtaaggc
     1741 cgactcgagt ccctatcagt gatagagatt gacatcccta tcagtgatag agatactgag
     1801 cacatcagca ggacgcactg accgaattca ttaaatttaa ctttaagaag gagatataca
     1861 tatgacctgg cttcccctta atcccattcc actcaaagat cgcgtctcca tgatctttct
     1921 gcaatatggg cacatcgatg taatagatgg cgcgtttgta cttatcgaca agacagggat
     1981 ccgcactcat attcctgttg gctcggttgc ctgcatcatg ctggaacctg gtacacgggt
     2041 ttcgcatgca gctgtacgcc tggctgcgca agttggaaca ttgttggtat gggtggggga
     2101 agcgggcgtt cgtgtttatg cttctggtca gcctggaggt gcgcgttcag ataagctgct
     2161 ctatcaggca aaacttgctc tggatgaaga tttgcgtctg aaggtcgtac gtaaaatgtt
     2221 tgaacttcgg tttggagaac ctgcgcctgc caggcgctcc gtagagcaac tcagaggtat
     2281 agaaggcagt cgcgtgcggg caacctacgc acttctggcg aagcaatacg gcgtgacatg
     2341 gaatggacgt cgctacgatc cgaaagactg ggaaaagggc gatacgatca accaatgcat
     2401 aagcgctgca acttcctgtt tatacggcgt aactgaagcg gcgatacttg cagctggtta
     2461 tgcacaagct attgggtttg tgcatacagg aaagcctctt tcctttgttt acgatattgc
     2521 agacatcatt aaatttgaca ctgttgtacc gaaagctttt gagatagcgc gtcgtaaccc
     2581 tgatgagccg gaccgggaag tccgtttggc gtgcagggat atttttcgca gtagtaaaac
     2641 attagccaaa ttgattccgc ttatagagga cgtgcttgcc gctggagata tacaaccgcc
     2701 ggccccacct gaagatgcac agcctgttgc cattccgctt cctgtttcac agggagatgc
     2761 aggtcatcgg agtagctgaa atgagtatgt tggtcgtggt cactgaaaat gtacctccgc
     2821 gcttacgagg cagattagcc atctggttgt tggaggtacg tgcaggggta tatgtaggtg
     2881 atgtatccgc aaaaattcgt gaaatgatct gggaacaaat agctggactg gcggaagaag
     2941 gcaatgtagt gatggcatgg gcaacgaata cggaaacggg atttgagttc cagacatttg
     3001 ggttaaacag gcgtaccccg gtagatttgg atggtttaag gttggtgtct tttttacctg
     3061 tttgagcctt aattaacggc actcctcagc aaatataatg accctcttga taacccaaga
     3121 gggcattttt taatgcccat ggcgtttacc acagctaaca ccacgtcgtc cctatctgct
     3181 gccctaggtc tatgagtggt tgctggataa ctttacgggc atgcataagg ctcgtaggct
     3241 atattcaggg agaccacaac ggtttccctc tacaaataat tttgtttaac tttgaaataa
     3301 ggaggtaata caaatgtctc gtttagataa aagtaaagtg attaacagcg cattagagct
     3361 gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt
     3421 agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc
     3481 cattgagatg ttagataggc accatactca cttttgccct ttagaagggg aaagctggca
     3541 agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg
     3601 agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca
     3661 attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc
     3721 tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga
     3781 agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt
     3841 atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg
     3901 attagaaaaa caacttaaat gtgaaagtgg gtcttaataa gcgacctcga gtctggtaaa
     3961 gaaaccgctg ctgcgaaatt tgaacgccag cacatggact cgtctactag cgcagcttaa
     4021 ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     4081 cgggtcttga ggggtttttt gctgaaacct caggcatttg agaagcacac ggtcacactg
     4141 cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc
     4201 tgccctgaac cgacgaccgg gtcatcgtgg ccggatcttg cggcccctcg gcttgaacga
     4261 attgttagac attatttgcc gactaccttg gtgatctcgc ctttcacgta gtggacaaat
     4321 tcttccaact gatctgcgcg cgaggccaag cgatcttctt cttgtccaag ataagcctgt
     4381 ctagcttcaa gtatgacggg ctgatactgg gccggcaggc gctccattgc ccagtcggca
     4441 gcgacatcct tcggcgcgat tttgccggtt actgcgctgt accaaatgcg ggacaacgta
     4501 agcactacat ttcgctcatc gccagcccag tcgggcggcg agttccatag cgttaaggtt
     4561 tcatttagcg cctcaaatag atcctgttca ggaaccggat caaagagttc ctccgccgct
     4621 ggacctacca aggcaacgct atgttctctt gcttttgtca gcaagatagc cagatcaatg
     4681 tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat tgcgctgcca ttctccaaat
     4741 tgcagttcgc gcttagctgg ataacgccac ggaatgatgt cgtcgtgcac aacaatggtg
     4801 acttctacag cgcggagaat ctcgctctct ccaggggaag ccgaagtttc caaaaggtcg
     4861 ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg tcaccgtaac cagcaaatca
     4921 atatcactgt gtggcttcag gccgccatcc actgcggagc cgtacaaatg tacggccagc
     4981 aacgtcggtt cgagatggcg ctcgatgacg ccaactacct ctgatagttg agtcgatact
     5041 tcggcgatca ccgcttccct catactcttc ctttttcaat attattgaag catttatcag
     5101 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaatagct
     5161 agctcactcg gtcgctacgc tccgggcgtg agactgcggc gggcgctgcg gacacataca
     5221 aagttaccca cagattccgt ggataagcag gggactaaca tgtgaggcaa aacagcaggg
     5281 ccgcgccggt ggcgtttttc cataggctcc gccctcctgc cagagttcac ataaacagac
     5341 gcttttccgg tgcatctgtg ggagccgtga ggctcaacca tgaatctgac agtacgggcg
     5401 aaacccgaca ggacttaaag atccccaccg tttccggcgg gtcgctccct cttgcgctct
     5461 cctgttccga ccctgccgtt taccggatac ctgttccgcc tttctccctt acgggaagtg
     5521 tggcgctttc tcatagctca cacactggta tctcggctcg gtgtaggtcg ttcgctccaa
     5581 gctgggctgt aagcaagaac tccccgttca gcccgactgc tgcgccttat ccggtaactg
     5641 ttcacttgag tccaacccgg aaaagcacgg taaaacgcca ctggcagcag ccattggtaa
     5701 ctgggagttc gcagaggatt tgtttagcta aacacgcggt tgctcttgaa gtgtgcgcca
     5761 aagtccggct acactggaag gacagatttg gttgctgtgc tctgcgaaag ccagttacca
     5821 cggttaagca gttccccaac tgacttaacc ttcgatcaaa ccacctcccc aggtggtttt
     5881 ttcgtttaca gggcaaaaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5941 cttttctact gaaccgctct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     6001 agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg
     6061 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     6121 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     6181 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6241 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     6301 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     6361 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     6421 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     6481 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     6541 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     6601 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     6661 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     6721 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     6781 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     6841 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     6901 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     6961 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     7021 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     7081 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     7141 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     7201 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     7261 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     7321 gacgctctcc cttatgcgac tcctgcatta ggaaat
//
