LOCUS       sequence-428839-        4555 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses non-target sequence for Haliangium type III CRISPR-Cas 
            complex; identical to pTarget (Addgene plasmid # 214052) but does 
            not contain a protospacer..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4555)
  AUTHORS   Steens JA, Bravo JPK, Salazar CRP, Yildiz C, Amieiro AM, Kostlbacher
            S, Prinsen SHP, Andres AS, Patinios C, Bardis A, Barendregt A, 
            Scheltema RA, Ettema TJG, van der Oost J, Taylor DW, Staals RHJ
  TITLE     Type III-B CRISPR-Cas cascade of proteolytic cleavages.
  JOURNAL   Science. 2024 Feb 2;383(6682):512-519. doi: 10.1126/science.adk0378.
            Epub 2024 Feb 1.
  PUBMED    38301007
REFERENCE   2  (bases 1 to 4555)
  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: 
            "10.1126/science.adk0378"; journalName: "Science"; date: "2024-02-2-
            2"; volume: "383"; issue: "6682"; pages: "512-519"
COMMENT     Sequence Label: pNon-target
FEATURES             Location/Qualifiers
     source          1..4555
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..23,24..30)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    38..54
                     /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     43..65
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    71..140
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             191..217
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             233..250
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             254..286
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(290..298,299..313)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(444..461)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      614..700
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      792..819
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(838..932)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(963..1757)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1074..1093)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1684..1703
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      2064..2652
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2553..2572
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2806..2823
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2838..2978
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2904..2923)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3064..3086
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3164..3241
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3242..4324
                     /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     complement(3261..3280)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcac
       61 acaggaaaca gcgccgctga gaaaaagcga agcggcactg ctctttaaca atttatcaga
      121 caatctgtgt gggcactcga ccggaattat cgattaactt tattattaaa aattaaagag
      181 gtatatatta atgtatcgat taaataagga ggaataaacc atggggggtt ctcatcatca
      241 tcatcatcat ggtatggcta gcatgactgg tggacagcaa atgggtcggg atctgtacga
      301 cgatgacgat aaggatccaa cccttttcca agcttcattg ctaacgctta tttagaagta
      361 gcgttagaat caagcaaagg cagcatgctt ccaagggcga attcgaagct tggctgtttt
      421 ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg
      481 ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac
      541 tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg
      601 aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
      661 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa
      721 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca
      781 tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctatt
      841 ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc
      901 tgaggtcatt actggatcta tcaacaggag tccaagcgag ctctcgaacc ccagagtccc
      961 gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg
     1021 ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca
     1081 cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg
     1141 aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc
     1201 acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc
     1261 gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga
     1321 gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca
     1381 agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg
     1441 tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
     1501 tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc
     1561 cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga
     1621 accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt
     1681 tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat
     1741 ccatcttgtt caatcatgcg aaacgatcct catcctgtct cttgatcaga tcatgatccc
     1801 ctgcgccatc agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca
     1861 accttaccag agggcgcccc agctggcaat tccctgtcag accaagttta ctcatatata
     1921 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     1981 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2041 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2101 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2161 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2221 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2281 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2341 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2401 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2461 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2521 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2581 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2641 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2701 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     2761 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     2821 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     2881 caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     2941 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     3001 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3061 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     3121 agatcaattc gcgcgcgaag gcgaagcggc atgcatttac gttgacacca tcgaatggtg
     3181 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     3241 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     3301 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     3361 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     3421 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     3481 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     3541 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     3601 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     3661 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     3721 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     3781 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     3841 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     3901 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     3961 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     4021 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     4081 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     4141 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     4201 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     4261 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     4321 gtgagcgcaa cgcaattaat gtaagttagc gcgaattgat ctggtttgac agcttatcat
     4381 cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc
     4441 tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat
     4501 aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctg
//
