LOCUS       sequence-227222-        7436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses RPCC7116RT-Cas1-Cas2 under pT7lac promoter, encodes 
            RPCC7116CRISPR array 2, compatible with SENECA acquisition readout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7436)
  AUTHORS   Schmidt F, Cherepkova MY, Platt RJ
  TITLE     Transcriptional recording by CRISPR spacer acquisition from RNA.
  JOURNAL   Nature. 2018 Oct;562(7727):380-385. doi: 10.1038/s41586-018-0569-1. 
            Epub 2018 Oct 3.
  PUBMED    30283135
REFERENCE   2  (bases 1 to 7436)
  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: 
            "10.1038/s41586-018-0569-1"; journalName: "Nature"; date: "2018-10";
            volume: "562"; issue: "7727"; pages: "380-385"
COMMENT     Sequence Label: 
            pFS_0387_pET30-RT-CRISPR_Rivularia_sp._PCC_7116_ARRAY_2
FEATURES             Location/Qualifiers
     source          1..7436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(210..1025)
                     /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(210..1025)
                     /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     933..952
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1017..1036
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1147..1735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1636..1655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1889..1906
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1921..2060
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1986..2005)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2146..2168
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2162..2353)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3162..4244)
                     /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     4206..4225
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4245..4322)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4528..4547
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4635..4654
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4635..4653
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4654..4678
                     /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)."
     RBS             4693..4715
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(7200..7218)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7214..7261
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ggccgctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
       61 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
      121 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
      181 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
      241 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
      301 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
      361 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
      421 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
      481 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
      541 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
      601 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
      661 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
      721 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
      781 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
      841 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
      901 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
      961 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     1021 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     1081 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     1141 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     1201 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     1261 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     1321 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     1381 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     1441 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     1501 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     1561 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     1621 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     1681 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     1741 cagcaacgcg gcctttttac ggttcctggt cttttgctgg ccttttgctc acatgttctt
     1801 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     1861 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     1921 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     1981 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     2041 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     2101 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     2161 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     2221 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     2281 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     2341 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     2401 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     2461 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     2521 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     2581 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     2641 gactttacga aacacggaaa ccgaagatca ttcatgttgt tgctcaggtc gcagacgttt
     2701 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     2761 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     2821 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     2881 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     2941 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     3001 gtcctacgag ttgcatgata aagaagatag tcataagtgc ggcgacgata gtcatgcccc
     3061 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     3121 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     3181 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3241 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     3301 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     3361 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct atcttcggta
     3421 tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg
     3481 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     3541 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     3601 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     3661 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     3721 accagatgct ccacgcccag tcgcgtacca tcttcatggg agaaaataat actgttgatg
     3781 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     3841 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     3901 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     3961 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     4021 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     4081 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     4141 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     4201 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     4261 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     4321 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     4381 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     4441 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     4501 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     4561 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct
     4621 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     4681 tctagaaata attttgttta actttaagaa ggagatatac atatgggata cgagaacatc
     4741 agataccttc acaagccgta cttcgcagaa acgattactg agaagccacc acagactgag
     4801 gaagatgact caccattcca gaagaacatc tggaacgctg agatggctgc aatctatctt
     4861 atcgagcagg gtacaaacat ctacaaagac taccagcgtt tcataataca cgttagtgag
     4921 aagccaaaac ttgaggtccc tatcagggac gtacaacaga tcatcgtctt tggaaatatc
     4981 cagctaagca cgcctgttat ccaggcctgc ctgaaggagc agatcccagt tgttttcctg
     5041 tctcagaccg gaacatacca tggccatctg tggtcggaga agtcaatcca ccttgataac
     5101 cagttggttc aggctgagcg aaggaacgac gacttattca gattctcagt tagccgagca
     5161 gtggttctag ggaagcttct gaactctaag caactactga tgagattcaa cagaaggaga
     5221 aagattggta aggtcgaaga ggcaatctac ggtatcaacc aggacataga cgcattgaat
     5281 tacgtggaca acctggacac gctgagggga tacgagggta tcgcagccgc ccgttacttc
     5341 cccgccttcg gtaacttgat aacaaaccca aagttcagct tcagtcaaag gttcaggcag
     5401 ccaccaactg acgaaattaa ctctctactg tccttcggtt acacattgtt attcaacaac
     5461 gttttatcct tcatcatcac tgagggcttg tcgccgtaca taggacactt ccattacggt
     5521 gacaagcaga aaacttacct cgcattcgac cttatggaag agttcagatc gcctatcgta
     5581 gactctcttg tattgaagat aattaacaag agcctattca agccccaaga ctttgacgtt
     5641 gtcgcctcta ccggcggtgt ttatttgtct cagaccagcc gtagggtgtt cctgaagcaa
     5701 ttcgagaaca gaatgaacga ggaaatctcc cacccggacc tgatctccca ggttacttac
     5761 cgtcatgcta tccagttgca ggtcagacga tacaaaaggt gcctgctgag tgacaacatc
     5821 tacgagtctt tcctaagggc tgactaatta actttaagaa ggagatatac atatgatgat
     5881 atcatctcag ttcatagaca taaacaactt ccagagagca tgggagaaag tcgcagacaa
     5941 gagagggtgt gctggagtag acggagagac aatctcctca ttcgccagta accagactgt
     6001 gaacgtctac caactcatga atagtgtggc agacggatct taccagccgt tcccatgtaa
     6061 acaagtcatc atcccaaaga ggaatgggtc ccagagggaa ctaaagatac cgaccatcag
     6121 ggataggata gtgcagcagg ctctcctaaa cgtcatctca cctcttatgg aagaaaagtt
     6181 ctctccagtc tctttcgcat acaggccaaa cttgagctac attaacgccg tggagaagat
     6241 cgcagattgg agagatatgg ggtacgtttg ggtcctggac gccgacattg tgaaattctt
     6301 cgacaacatc gatcaccaca gacttttgca gcaagtgcga ctacacatcg atcaccctgg
     6361 catcctgtgc ctgataaagg catggatctc tgtcggcgtc gagacacgag agggtttgat
     6421 tcttccacag aaaggaatcc cacagggcgc tgtcatctca cctatcttag ctaacatcta
     6481 cctgcacgag ttcgacgaga taatctctgc atctgaccta gagatagtaa gatacgccga
     6541 cgacttctta gtactatcca ccagccagga gagaatagcc atagccaagt cccaggttat
     6601 agacttgctc gactcattgg gtctagagat taacacagac aagacacaga tcacgtcttt
     6661 cgagagagga ttcagattcc ttggacacgg gtttctgtcc gacgcaatat ttccagtgga
     6721 caccaacaag gctaaactga aaagtggaat cgagacaaac agagaaaaga ctaggacccg
     6781 aaagacgagc aagaagaagc tctaccataa tccttacaga aataagaagg tcgtatgacg
     6841 tacgttaact ttaagaagga gaacttaaga tgcttgtggt ggtggtttat gacattccca
     6901 atgacaagag gcgaacaaaa ctgtctaact tcttggaagg gtacggacag cgtgtgcagt
     6961 tctcagtttt cgagtgcttc ttaaacctgg acgaaatgag acagctattc gagaaaagca
     7021 agaagattgt aaagccgtct gaggataacg tgagattcta ctggatatca gaggacgcag
     7081 tgtctcgtgt gcttaccata ggctctgagc accccaatgc cccacctaat tattacgtca
     7141 tctaactcga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     7201 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     7261 gctgaaagga ggaactatat ccggatacca ggtaaagcag aagtggaagc actttgtggc
     7321 agtacgcttg tggaagtact tcttggcagt tactgccatc ttctcgttca agttacaaga
     7381 tgctcggttg aaaccccacc gattgggtta attcggatta gttggaaacg tcccgc
//
