LOCUS       sequence-489586-        6659 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  for E.coli plasmid interference and PAM depletion assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6659)
  AUTHORS   Jin S, Zhu Z, Li Y, Zhang S, Liu Y, Li D, Li Y, Luo Y, Cheng Z, Zhao
            KT, Gao Q, Yang G, Li H, Liang R, Zhang R, Qiu JL, Zhang YE, Liu JG,
            Gao C
  TITLE     Functional RNA splitting drove the evolutionary emergence of type V 
            CRISPR-Cas systems from transposons.
  JOURNAL   Cell. 2025 Sep 29:S0092-8674(25)01035-9. doi: 
            10.1016/j.cell.2025.09.004.
  PUBMED    41027434
REFERENCE   2  (bases 1 to 6659)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2025 
            Sep 29:S0092-8674(25)01035-9. doi: 10.1016/j.cell.2025.09.004."
COMMENT     Sequence Label: pEffector-LaTranC-CRISPR RNA
FEATURES             Location/Qualifiers
     source          1..6659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(52..867)
                     /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"
     primer_bind     775..794
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     859..878
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1258..1275)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1392..1937)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(1472..1491)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2013..2032
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2186..2220
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     CDS             3047..3076
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3092..3109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3165..3182)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3335..3381
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3408..4490)
                     /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     4452..4471
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4625..4630,4631..4646,4647..4653)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4659..4678
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     misc_RNA        4681..4730
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
ORIGIN
        1 cagataaaaa aaatccttag ctttcgctaa ggatgatttc tgctagtatt attagaaaaa
       61 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
      121 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc aaagaatggc
      181 aaggtcctgg taacggtctg cgattccgac ccgtccaaca tcaatacaac ctattaattt
      241 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
      301 tgagaatggc aagagcttgt gcatttcttt ccagacttgt tcaacaggcc agccattacg
      361 ctcgtcatca aaatcactcg catcaaccaa accgttattc atgcgtgatt gcgcctgagc
      421 aagacgaaat acacgatcgc tgttaaaagg acaattacaa acaggaatcg aatgtaaccg
      481 gcgcaggaac acggccagcg catcaacaat attttcacct gaatcaggat attcttctaa
      541 tacctggaag gctgttttcc caggaatcgc ggtggtgagt aaccacgcat catcaggagt
      601 acggataaaa tgcttgatgg tcgggagagg cataaactcc gtcagccagt tgagacggac
      661 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
      721 cgcatcgggc ttcccataca agcgatagat tgtcgcacct gattgcccga cattatcgcg
      781 agcccattta tacccatata aatcagcgtc catgttggag tttaagcgcg gacgggagca
      841 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
      901 cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg
      961 agacacaacg tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagctcta
     1021 gtagttacat tgtcgatctg ttcatggtga acagctttga atgcaccaaa aactcgtaaa
     1081 agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatagcta gcggagtgta
     1141 tactggctta ctatgttggc actgatgagg gtgtcagtga agtgcttcat gtggcaggag
     1201 aaaaaaggct gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc
     1261 gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg
     1321 gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg
     1381 gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca
     1441 aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc
     1501 tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg
     1561 ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct
     1621 ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg
     1681 tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg
     1741 atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt
     1801 ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa
     1861 ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca
     1921 gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc taggatcatg
     1981 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     2041 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctgctca
     2101 tgtttgacag cttatcatcg atgcataatg tgcctgtcaa atggacgaag cagggattct
     2161 gcaaacccta tgctactccc acgagttgac agctagctca gtcctaggta taatactagt
     2221 gaagcacttc acagaagtgc atggagcaga aggtgatcga cagcaacaag tgagcggacg
     2281 caactagaag cacttcacag aagtgcatgg agcagaagcg cactccgtct ctgccctgtc
     2341 aaggcagctg ttttgcaagt tcgattatac ctttggggta aatggttgtc aatggcgggc
     2401 ccgcgacaga aaataggaag cctttctctg cacattcaac aaacaacatt tacagaatgc
     2461 cccaattatg ttatactaat gttgccttgc aataatgtga acgtgaaatg gagggatgtc
     2521 tgtggcagat atcaaatctc aaagtggtgg gaggtctgtc cccaccatgg ggtgcgaacc
     2581 ttgtgtgctc atcattgccg tgagcgttcg cacgtccaaa cgaccatatc atcgttgccc
     2641 tgcgaccatt cagcggcaat caagacgcag gcatgatatg taaccatgca tttctgtgaa
     2701 ctgcttccaa aacgcactgc ttcattataa tcctccctgt gcagttctgc cgaagcactt
     2761 cacagaagtg catggagcag aaggtgatcg acagcaacaa gtgagcggac gcaactagaa
     2821 gcacttcaca gaagtgcatg gagcagaaga agtcgtttat agcggtttgt ttacagggag
     2881 ttagaagcac atttcaattt ttcagtaaaa ctcctccagc tttgcagctg ggggagtttt
     2941 ttactcccct cttgacaatt atcttctacg tctgtagaat ataatcaaac tacaaccgta
     3001 gaagatattc acagaaggga ggataacgct atggaagctt gggcccgaac aaaaactcat
     3061 ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttaaac
     3121 ggtctacagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
     3181 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
     3241 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     3301 tctacccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     3361 agactgggcc tttcgtttta tctgttgttt gtcggtgaac tgaattctca ctgcccgctt
     3421 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     3481 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac tggcaacagc
     3541 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc
     3601 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctatct
     3661 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta
     3721 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg
     3781 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct
     3841 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga
     3901 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc
     3961 aatgcgacca gatgctccac gcccagtcgc gtaccgtcct catgggagaa aataatactg
     4021 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct
     4081 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt
     4141 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc
     4201 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc
     4261 gacggcgcgt gcagggccag actggaggtg gcaactccaa tcagcaacga ctgtttgccc
     4321 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact
     4381 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga
     4441 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcat attcaccacc
     4501 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg
     4561 atggcgcgcc gcttcgtcag gccacatagc tttcttgttc tgatcggaac gatcgttggc
     4621 tgtgttgaca attaatcatc ggctcgtata atgtgtggaa ttgtgagcgc tcacaattag
     4681 ctgtcaccgg atgtgctttc cggtctgatg agtccgtgag gacgaaacag cctctacaaa
     4741 taattttgtt taaagatcta aaataaggag gaaaaaaaaa tgagtgtatc tttttcacta
     4801 aatgctaaaa agattcgcct tgagaactac gcaatgaaaa tgcgtctgta tccgtcgccg
     4861 acccaggcag agcagatgga caaaatgttc ctggcgctgc gtttagcgta taacatgacc
     4921 ttccacgaag tttttcagca aaacccggcg gtgtgtggcg acccggacga ggatggcaat
     4981 gtgtggccta gctataaaaa gatggcaaat aagacctggc gtaaagcgct aattgatcag
     5041 aacccggcga tcgccgaggc gccagcagcg gcgatcacca ccaataacgg tctgtttctc
     5101 tctaatggtc agaaggcgtg gaaaactggt atgcacaatc tgccggctaa caaggcggat
     5161 cgcaaagact tccgcttcta cagcttgtcg aaaccgcgtc gcagcttcgc ggtccagatc
     5221 ccaccggact gtattatccc gagcgacacg aaccagaagg tggcccgtat caaactgccg
     5281 aaaattgatg gtgctattaa ggctcgtggt tttaatcgta agatttggtt tggtccggat
     5341 ggcaagcaca cctacgagga agccctggct gcacacgaac tgtctaacaa cttaaccgtt
     5401 cgtgtttcca aggacacgtg cggcgactac ttcatctgca ttaccttcag ccagggtaaa
     5461 gtgaaaggtg ataaaccgac ctgggaattt tatcaagaag tgcgtgtgtc cccgatcccg
     5521 gagccgattg gtctggatgt gggcattaag gacatcgcga tcctgaatac cggcacgaaa
     5581 tacgaaaaca aacagtttaa acgtgatcgt gctgcgacct tgaaaaagat gagccgccaa
     5641 ctgtcccgtc gctggggtcc ggcaaacagc gcgttccgcg actataataa gaacatccgc
     5701 gcggaaaacc gtgcgctgga gaaggcgcaa caagatccgg gctcatctgg tgttggccca
     5761 gaggcgccgg tgctgaagag cgtggcccag ccgtctcgtc gctacctgac cattcagaag
     5821 aaccgtgcca agttggaacg taaaatcgct agacgtaggg acacttatta ccatcaggtt
     5881 actgctgaag ttgcaggtaa atcctcgctt ctggccgtgg aaaccctgcg tgtcaagaac
     5941 atgcttcaaa atcatcgcct cgccttcgcg ctgagcgacg cagcaatgtc cgatttcatt
     6001 tccaaactga agtacaaggc gagacgcatc caagtaccgc tggttgccat cggcacgttc
     6061 cagccttcta gccaaacctg tagcgtttgc ggaagcatca acccggcagt caagaacctg
     6121 tctatcagag tttggacctg cccgaattgc ggcactcgcc ataaccgcga tattaacgca
     6181 gcgaagaaca ttctggcgat cgctcaaaac atgttggaga aaaaggtgcc atttgcggat
     6241 gaggcgttgc cagacgagaa acctccggcg gcgccggtga agaaggccgc ccgtaaaccg
     6301 cgtgatgcag tctttccgga ccacccggac ctggtgatcc gttttagcaa agagttgacg
     6361 cagctgaatg acccgcgtta cgttatcgtt aataaggcaa ccaatcagat agtggataac
     6421 gcccaaggcg ctggttatcg tagcgctgcc aaggcgaaga actgctataa agcgaaattg
     6481 gcgtggagca gtaagaccaa taagtaactc gaggagctca aataaagccc tgagtttaac
     6541 cgctcggggc tttttgcgtt tctgagtagg acaaatccgc cgggagcgga tttgaacgtt
     6601 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatca
//
