LOCUS       sequence-150459-        5032 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes Cas1 and Cas2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5032)
  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 5032)
  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: pRSF-DUET Cas1 Cas2
FEATURES             Location/Qualifiers
     source          1..5032
                     /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             1001..1018
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1132..1150
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1151..1175
                     /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             1569..1613
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1651..1669)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1665..1712
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1942..2757)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1942..2757)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2665..2684
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2758..2849)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2865..3614)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(2875..2892)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3115..3134)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3810..4892)
                     /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     4854..4873
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4893..4970)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(5016..5032,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatataca tgacctggct tccccttaat cccattccac tcaaagatcg cgtctccatg
      121 atctttctgc aatatgggca gatcgatgta atagatggcg cgtttgtact tatcgacaag
      181 acagggatcc gcactcatat tcctgttggc tcggttgcct gcatcatgct ggaacctggt
      241 acacgggttt cgcatgcagc tgtacgcctg gctgcgcaag ttggaacatt gttggtatgg
      301 gtgggggaag cgggcgttcg tgtttatgct tctggtcagc ctggaggtgc gcgttcagat
      361 aagctgctct atcaggcaaa acttgctctg gatgaagatt tgcgtctgaa ggtcgtacgt
      421 aaaatgtttg aacttcggtt tggagaacct gcgcctgccc ggcgctccgt agagcaactc
      481 agaggtatag aaggcagtcg cgtgcgggca acctacgcac ttctggcgaa gcaatacggc
      541 gtgacatgga atggacgtcg ctacgatccg aaagactggg aaaagggcga tacgatcaac
      601 caatgcatta gcgctgcaac ttcctgttta tacggcgtaa ctgaagcggc gatacttgca
      661 gctggttatg caccagctat tgggtttgtg catacaggaa agcctctttc ctttgtttac
      721 gatattgcag acatcattaa atttgacact gttgtaccga aagcttttga gatagcgcgt
      781 cgtaaccctg gtgagccgga ccgggaagtc cgtttggcgt gcagggatat ttttcgcagt
      841 agtaaaacat tagccaaatt gattccgctt atagaggacg tgcttgccgc tggagaaata
      901 caaccgccgg ccccacctga agatgcacag cctgttgcca ttccgcttcc tgtttcactg
      961 ggagatgcag gccatcggag tagctgacat gggcagcagc catcaccatc atcaccacag
     1021 ccaggatccg aattcgagct cggcgcgcct gcaggtcgac aagcttgcgg ccgcataatg
     1081 cttaagtcga acagaaagta atcgtattgt acacggccgc ataatcgaaa ttaatacgac
     1141 tcactatagg ggaattgtga gcggataaca attccccatc ttagtatatt agttaagtat
     1201 aagaaggaga tatacaatga gtatgttggt cgtggtcact gaaaatgtac ctccgcgctt
     1261 acgaggcaga ttagccatct ggttgttgga ggtacgtgca ggggtatatg taggtgatgt
     1321 atccgcaaaa attcgtgaaa tgatctggga acaaatagct ggactggcgg aagaaggcaa
     1381 tgtagtgatg gcatgggcaa cgaatacgga aacgggattt gagttccaga catttgggtt
     1441 aaacaggcgt accccggtag atttggatgg tttaaggttg gtgtcttttt tacctgtttg
     1501 atatggcaga tctcaattgg atatcggccg gccacgcgat cgctgacgtc ggtaccctcg
     1561 agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta
     1621 gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg
     1681 gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca
     1741 cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta
     1801 tttaacgacc ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc aagtggcact
     1861 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1921 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     1981 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2041 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2101 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2161 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2221 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     2281 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg
     2341 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     2401 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     2461 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     2521 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     2581 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     2641 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     2701 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcatact
     2761 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2821 atttgaatgt atttagaaaa ataaacaaat aggcatgcag cgctcttccg cttcctcgct
     2881 cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gtgtcagctc actcaaaagc
     2941 ggtaatacgg ttatccacag aatcagggga taaagccgga aagaacatgt gagcaaaaag
     3001 caaagcaccg gaagaagcca acgccgcagg cgtttttcca taggctccgc ccccctgacg
     3061 agcatcacaa aaatcgacgc tcaagccaga ggtggcgaaa cccgacagga ctataaagat
     3121 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3181 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     3241 gttggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     3301 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     3361 gacacgactt atcgccactg gcagcagcca ttggtaactg atttagagga ctttgtcttg
     3421 aagttatgca cctgttaagg ctaaactgaa agaacagatt ttggtgagtg cggtcctcca
     3481 acccacttac cttggttcaa agagttggta gctcagcgaa ccttgagaaa accaccgttg
     3541 gtagcggtgg tttttcttta tttatgagat gatgaatcaa tcggtctatc aagtcaacga
     3601 acagctattc cgttactcta gatttcagtg caatttatct cttcaaatgt agcacctgaa
     3661 gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga
     3721 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     3781 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3841 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     3901 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     3961 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     4021 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     4081 actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     4141 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     4201 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     4261 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     4321 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     4381 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     4441 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     4501 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     4561 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     4621 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     4681 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     4741 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     4801 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     4861 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     4921 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     4981 acgctctccc ttatgcgact cctgcattag gaaattaata cgactcacta ta
//
