LOCUS       sequence-428822-        4551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses target sequence for Haliangium type III CRISPR-Cas 
            complex; identical to pNon-target (Addgene plasmid # 214053) but 
            contains a protospacer..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4551)
  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 4551)
  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: pTarget
FEATURES             Location/Qualifiers
     source          1..4551
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..795)
                     /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(112..131)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     722..741
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      1102..1690
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1591..1610
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1844..1861
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1876..2016
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1942..1961)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2102..2124
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2202..2279
                     /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             2280..3362
                     /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(2299..2318)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3594..3599,3600..3616,3617..3623)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3631..3647
                     /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     3636..3658
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    3664..3733
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             3784..3810
                     /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             3826..3843
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3847..3879
                     /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(3883..3891,3892..3906)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     primer_bind     complement(4033..4050)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4203..4289
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4381..4408
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4427..4521)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
       61 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
      121 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
      181 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
      241 gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc
      301 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
      361 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
      421 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
      481 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
      541 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
      601 cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
      661 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
      721 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
      781 atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc atgatcccct
      841 gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac
      901 cttaccagag ggcgccccag ctggcaattc cctgtcagac caagtttact catatatact
      961 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     1021 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1081 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     1141 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     1201 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     1261 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     1321 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     1381 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     1441 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     1501 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     1561 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     1621 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1681 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1741 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     1801 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     1861 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     1921 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     1981 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     2041 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     2101 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag
     2161 atcaattcgc gcgcgaaggc gaagcggcat gcatttacgt tgacaccatc gaatggtgca
     2221 aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg
     2281 tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt
     2341 cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg
     2401 cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt
     2461 cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg
     2521 cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac
     2581 gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg
     2641 ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca
     2701 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt
     2761 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
     2821 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
     2881 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
     2941 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
     3001 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
     3061 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
     3121 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
     3181 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
     3241 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
     3301 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     3361 gagcgcaacg caattaatgt aagttagcgc gaattgatct ggtttgacag cttatcatcg
     3421 actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg
     3481 tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa
     3541 tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa
     3601 ttaatcatcc ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     3661 acagcgccgc tgagaaaaag cgaagcggca ctgctcttta acaatttatc agacaatctg
     3721 tgtgggcact cgaccggaat tatcgattaa ctttattatt aaaaattaaa gaggtatata
     3781 ttaatgtatc gattaaataa ggaggaataa accatggggg gttctcatca tcatcatcat
     3841 catggtatgg ctagcatgac tggtggacag caaatgggtc gggatctgta cgacgatgac
     3901 gataaggatc caaccctttt ccaagctttc cgagggagtt gaagcttggt tcaaagaacg
     3961 tatcaagagc agcatgcttc caagggcgaa ttcgaagctt ggctgttttg gcggatgaga
     4021 gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa
     4081 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
     4141 acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc
     4201 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     4261 cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc
     4321 aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc
     4381 agaaggccat cctgacggat ggcctttttg cgtttctaca aactctattc tcaccaataa
     4441 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta
     4501 ctggatctat caacaggagt ccaagcgagc tctcgaaccc cagagtcccg c
//
