LOCUS       sequence-150473-        6068 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encodes Cas1 P294H and Cas2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6068)
  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 6068)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pWUKI Cas1 P294H Cas2
FEATURES             Location/Qualifiers
     source          1..6068
                     /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)."
     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"
     protein_bind    1557..1578
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1593..1598,1599..1616,1617..1623)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1631..1647
                     /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     1636..1658
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1682..1969
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated and modified)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="modified to prevent cleavage by eukaryotic SUMO 
                     proteases"
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             2080..2892
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="SHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
                     PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKT
                     AFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDAS
                     DFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIA
                     DRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2080..2892
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="SHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
                     PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKT
                     AFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDAS
                     DFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIA
                     DRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2150..2169)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     CDS             complement(2984..3775)
                     /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(3776..3867)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3915..4653)
                     /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(4863..5945)
                     /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     5907..5926
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5946..6023)
                     /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 ccgcttcatg 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 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 gtgagcgcaa cgcaattaat
     1561 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     1621 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatgg ggtccctgca
     1681 ggactcagaa gtcaatcaag aagctaagcc agaggtcaag ccagaagtca agcctgagac
     1741 tcacatcaat ttaaaggtgt ccgatggatc ttcagagatc ttcttcaaga tcaaaaagac
     1801 cactccttta agaaggctga tggaagcgtt cgctaaaaga cagggtaagg aaatggactc
     1861 cttaagattc ttgtacgacg gtattagaat tcaagctgat caggcccctg aagatttgga
     1921 catggaggat aacgatatta ttgaggctca ccgcgaacag attggaggtc ggtttatccc
     1981 cgctggcgcg gggaactctc taaaagtata catttgttct taaagcattt tttcccatac
     2041 aaacaaccca ccaaccttaa tgtaacagga tctggcagta gccatattca acgggaaacg
     2101 tcttgctcta ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     2161 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     2221 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     2281 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     2341 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag
     2401 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     2461 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgaccg cgtatttcgt
     2521 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     2581 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc
     2641 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     2701 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     2761 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     2821 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     2881 gagtttttct aataaccgct atttaacgac cctgccctga accgacgacc gggtcatcgt
     2941 ggccggatct tgcggcccct cggcttgaac gaattgttag acattatttg ccgactacct
     3001 tggtgatctc gcctttcacg tagtggacaa attcttccaa ctgatctgcg cgcgaggcca
     3061 agcgatcttc ttcttgtcca agataagcct gtctagcttc aagtatgacg ggctgatact
     3121 gggccggcag gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg attttgccgg
     3181 ttactgcgct gtaccaaatg cgggacaacg taagcactac atttcgctca tcgccagccc
     3241 agtcgggcgg cgagttccat agcgttaagg tttcatttag cgcctcaaat agatcctgtt
     3301 caggaaccgg atcaaagagt tcctccgccg ctggacctac caaggcaacg ctatgttctc
     3361 ttgcttttgt cagcaagata gccagatcaa tgtcgatcgt ggctggctcg aagatacctg
     3421 caagaatgtc attgcgctgc cattctccaa attgcagttc gcgcttagct ggataacgcc
     3481 acggaatgat gtcgtcgtgc acaacaatgg tgacttctac agcgcggaga atctcgctct
     3541 ctccagggga agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat
     3601 caagccttac ggtcaccgta accagcaaat caatatcact gtgtggcttc aggccgccat
     3661 ccactgcgga gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga
     3721 cgccaactac ctctgatagt tgagtcgata cttcggcgat caccgcttcc ctcatactct
     3781 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     3841 ttgaatgtat ttagaaaaat aaacaaatag ctagctcact cggtcgctac gctccgggcg
     3901 tgagactgcg gcgggcgctg cggacacata caaagttacc cacagattcc gtggataagc
     3961 aggggactaa catgtgaggc aaaacagcag ggccgcgccg gtggcgtttt tccataggct
     4021 ccgccctcct gccagagttc acataaacag acgcttttcc ggtgcatctg tgggagccgt
     4081 gaggctcaac catgaatctg acagtacggg cgaaacccga caggacttaa agatccccac
     4141 cgtttccggc gggtcgctcc ctcttgcgct ctcctgttcc gaccctgccg tttaccggat
     4201 acctgttccg cctttctccc ttacgggaag tgtggcgctt tctcatagct cacacactgg
     4261 tatctcggct cggtgtaggt cgttcgctcc aagctgggct gtaagcaaga actccccgtt
     4321 cagcccgact gctgcgcctt atccggtaac tgttcacttg agtccaaccc ggaaaagcac
     4381 ggtaaaacgc cactggcagc agccattggt aactgggagt tcgcagagga tttgtttagc
     4441 taaacacgcg gttgctcttg aagtgtgcgc caaagtccgg ctacactgga aggacagatt
     4501 tggttgctgt gctctgcgaa agccagttac cacggttaag cagttcccca actgacttaa
     4561 ccttcgatca aaccacctcc ccaggtggtt ttttcgttta cagggcaaaa gattacgcgc
     4621 agaaaaaaag gatctcaaga agatcctttg atcttttcta ctgaaccgct ctagatttca
     4681 gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata taagttgtaa
     4741 ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     4801 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     4861 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4921 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     4981 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5041 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5101 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc
     5161 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5221 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5281 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5341 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5401 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     5461 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     5521 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     5581 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     5641 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     5701 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     5761 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     5821 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     5881 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     5941 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     6001 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6061 taggaaat
//
