LOCUS       sequence-182827-        5253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid containing the 3' portion of the ura4 marker and the gRNA 
            elements downstream of the target sequence. It serves as the PCR 
            template for generating the gRNA insert in the split-ura4 system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5253)
  AUTHORS   Zhang XR, He JB, Wang YZ, Du LL
  TITLE     A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in 
            Fission Yeast.
  JOURNAL   G3 (Bethesda). 2018 Apr 27. pii: g3.118.200164. doi: 
            10.1534/g3.118.200164.
  PUBMED    29703785
REFERENCE   2  (bases 1 to 5253)
  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: "G3 
            (Bethesda). 2018 Apr 27. pii: g3.118.200164. doi: 
            10.1534/g3.118.200164."
COMMENT     Sequence Label: pDB4282
FEATURES             Location/Qualifiers
     source          1..5253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(1819..2613)
                     /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(1930..1949)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2540..2559
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(2957..3385)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3067..3088)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3279..3298
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3526..3542
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3549..3568
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3549..3567
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     4750..4766
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(4816..4867)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     primer_bind     complement(5033..5049)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5033..5049)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5046..5068)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5057..5073
                     /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)."
     promoter        complement(join(5081..5087,5088..5105,5106..5111))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5126..5147
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaattc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     1861 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag
     1921 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag
     1981 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca
     2041 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc
     2101 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag
     2161 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg
     2221 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt
     2281 ctcggcagga gcaaggtggg atgacaggag atcctgcccc ggcacttcgc ccaatagcag
     2341 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt
     2401 ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc
     2461 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga
     2521 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg
     2581 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg
     2641 atcagatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac
     2701 tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc
     2761 tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt
     2821 tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg
     2881 tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc agcccttgcg
     2941 ccctgaattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3001 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3061 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3121 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3181 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3241 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3301 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3361 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     3421 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     3481 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     3541 gtgaattgta atacgactca ctatagggcg aattgggccc tctagatgca tgctcgagcg
     3601 gccgccagtg tgatggatat ctgcagaatt gcccttcatc tggtgtgtac aaaattgctt
     3661 cttgggctca tatcacaaat tgccatacag tgccaggcga gggtattata caaggcctca
     3721 aagaagttgg tttacctttg ggacgtggtc tcttgctttt ggctgaaatg tcttccaaag
     3781 gctctttggc tactggttcc tacacagaga aaaccttaga atggtttgag aagcataccg
     3841 atttttgctt tggctttata gctggtcgtc gatttcctaa ccttcaaagc gactacataa
     3901 ctatgtcccc tggtatcggc ttggatgtta aaggagacgg gctgggacag caatatcgta
     3961 ctcctgaaga agtgattgta aactgcggta gcgatatcat cattgttggt cgtggagtct
     4021 atggagctgg tcgtaatcct gttgtcgaag ccaagagata tagagaagct ggttggaagg
     4081 catatcagca aagactttct cagcattaaa aaaagactaa tgtaaaattt ttttggttgg
     4141 ttattgaaaa agtcgatgcc ttgtttgcgt ttgttttcct aggcgtttta tgtcagaagg
     4201 catttagaat tagtatacaa gtactctttg gtaaaatttt atgtagcgac taaaatatta
     4261 actattatag ataaacacct tgggaataaa aagtaatttg ctatagtaat ttattaaaca
     4321 tgctcctaca acattaccac aatcttttct cttggattga cattgaataa gaaaagagtg
     4381 aattttttta gacttgtaat gataactatg tacaaagcca atgaaagatg tatgtagatg
     4441 aatgtaaaat accatgtaga caaacaagat aaaacttggt tataaacatt ggtgttggaa
     4501 cagaataaat tagatgtcaa aaagtttcgt caatatcaca agctcatcgc gatggtatgc
     4561 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat
     4621 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     4681 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     4741 cacgacgttg taaaacgacg gccagtgcca agcttgcatg cctgcaggtc gactctagag
     4801 gatccccacg gtacccctgt ttcgtcctca cggactcatc agaccggaaa gcacatccgg
     4861 tgacagggca ccaccgactc ggtgccactc acttttttca agttgataac ggactagcct
     4921 tattttaact tgctatttct agctctaaaa cgcggccgct aagggcaatt ccagcacact
     4981 ggcggccgtt actagtggat ccgagctcgg taccaagctt ggcgtaatca tggtcatagc
     5041 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5101 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5161 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5221 gcgcggggag aggcggtttg cgtattgggc gct
//
