LOCUS       sequence-147777-        8367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for S. pombe. Allow sequence of interest 
            to be fused to N-terminal double V5 epitope tag.attR1-CmR-ccdB-attR2
            cassette, nmt1** (AT) promoter, ura4+ selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8367)
  TITLE     Akusjarvi and Bjerling plasmids
REFERENCE   2  (bases 1 to 8367)
  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"
COMMENT     Sequence Label: pREP82xV5_ccdB
FEATURES             Location/Qualifiers
     source          1..8367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        49..1200
                     /label=nmt1 P81 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring weak thiamine-repressible expression"
     CDS             1216..1257
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1264..1305
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    1315..1439
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1464..1469,1470..1487,1488..1494)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1548..2207
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1614..1633)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2549..2854
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2733..2752
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2895..3019)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     rep_origin      4124..4907
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(5257..5274)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5257..5273)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5266..5288)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5486..5941
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5573..5592)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5783..5804
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5958..5977)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6077..6099
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6137..6155)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6223..6327
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6328..6396,6397..7188)
                     /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     complement(6546..6565)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7359..7947
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7848..7867
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8101..8118
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8235..8256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8271..8276,8277..8294,8295..8301)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8309..8325
                     /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     8314..8336
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8333..8349
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8333..8349
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aagcttgcat gcctgcaggt cgatcgactc tagaggatca gaaaattatc gccataaaag
       61 acagaataag tcatcagcgg ttgtttcatt tcctatattt tttttttatt tttttatttt
      121 ttaataaggg aaaatttaac gtctaaggat acagaagatt gttagcacat taaagtaata
      181 aaggcttaag tagtaagtgc cttagcatgt tattgtattt caaaggacat aatctaaaat
      241 aataacaata tcatttctca caagttattc aattttcttt tttttttcta ataatatcaa
      301 gaatgtatta tttgtttgac ataagtcaac taatttattt aatatgctgg attaatcttg
      361 cagacatgta aattaacaag ttttagtcaa ataacgttga agtttcaatg aactcaaata
      421 atttctcttt ttttttatat aactctaatc tgatttatat tttccgcagg gatcaactga
      481 agttatgaca tttggattgg atcacttata accttggtcg ccaaataata caaaaatcag
      541 cgttataaaa caaagaaggt ttttgttaag aaattaatcc tctttcttga taagaaagtt
      601 gaaccgaaat tgcagatact gatatatgaa aataataccc acaattttgg gaatagcgca
      661 agcctcaatt taaacaatag gtgaggacac atgataatga cctcaatgat tgttagaaga
      721 aaagagcctc attacaaaat cgaaaaatga atggttgggt acaagtttcc aaaacatggt
      781 aaagtggact ttgcgtatga gacgtaaata gaaaaaaaca cttgttatat gttttctaga
      841 attattgttg tctctttatg gttggatgat gcaaaatagt aatttcggtt agttgctgta
      901 aaacaccacg agacaaatag atatggatat ttattaaatc aggaaaaacg taactctcgg
      961 ctactggatg gttcagtcac ccaacgatta ctggggagag aaaacagggc aaaagcaaag
     1021 cttaaaggaa tccgattgtc attcggcaat gtgcagcgaa actaaaaacc ggataatgga
     1081 cctgttaatc gaaacattga agatggaaga ggaatcctgg catatcatca attgaataag
     1141 ttgaattaat tatttcaatc tcattctcac tttctgactt atagtcgctt tgttaaatca
     1201 tacctcgagt ctatgggtaa acctattcct aaccctttgt taggcttgga cagtactggt
     1261 gctggtaagc ctattcccaa tcccttgctt ggtcttgact ctactggtca tatgacaagt
     1321 ttgtacaaaa aagctgaacg agaaacgtaa aatgatataa atatcaatat attaaattag
     1381 attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgg
     1441 cggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat aatgtgtgga
     1501 ttttgagtta ggatccgtcg agattttcag gagctaagga agctaaaatg gagaaaaaaa
     1561 tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat
     1621 ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt
     1681 taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc
     1741 gcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat
     1801 gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc
     1861 tctggagtga ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg
     1921 cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg
     1981 tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca
     2041 acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga
     2101 tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc
     2161 ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaatct agaggatccg
     2221 gcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa
     2281 tatatactga tatgtatacc cgaagtatgt caaaaagagg tatgctatga agcagcgtat
     2341 tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc
     2401 tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg
     2461 aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat gaacggctct
     2521 tttgctgacg agaacagggg ctggtgaaat gcagtttaag gtttacacct ataaaagaga
     2581 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg
     2641 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta
     2701 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt
     2761 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
     2821 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta tacacagcca
     2881 gtctgcaggt cgaccatagt gactggatat gttgtgtttt acagtattat gtagtctgtt
     2941 ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt tctcgttcag
     3001 ctttcttgta caaagtggtc tcgagcccgg gcggccgcta actaactaag ggtaaaagga
     3061 atgtctccct tgccagtact gctagggttt ttctttcaaa ctatggaagc ccattcaagc
     3121 tgcatattac gattttgttt ttcgctttta gaaagtggtt tagatgagat aatagaaaaa
     3181 ttcttgatct ccgacaacga gtacttttat tttttttgct aatcacttta ctcaatatta
     3241 gctcgaaatc gtagaaacgt agacgggtgc gggataccga gtggtgtagt taagaatttt
     3301 tataaaccac gtggcccaaa aatatgaacc caaaacgttt atacatgagt atactttaag
     3361 aaggctatac cccttcgtgt tagatgtagt tttagctacc caacccgagt ctatgagctt
     3421 gacttcagat gtagaaggca ttaaatcgtt ttgaatatta attaaaaaac gatgaaaatt
     3481 aaatatttaa aagcaatcat acgctgaaaa tttagtgctg tggctaatcc ttcaacatgg
     3541 aaatgccata aaagtgactt tgacaaaaaa aaaagtatat acaggtagta aactcatcta
     3601 cttcattgac tttgtttaca gcatgtggaa ggaggaatat ttattgctaa atcgtagttt
     3661 aacattcaat aagtaatact attgaaattc gacaagattg gccgcatgga tgaaaaagag
     3721 gcattttgct ttgggagaat tagttcaaat tagaactgaa aaaaaaaact ttacgaggca
     3781 aaaatgtcgg attgagatcg taaaagttcg ctcgtcgtct tttgctttgt gattgttttc
     3841 atggatacat cttgctggat atttaaattt tagtactatg tataagatat tctataaatg
     3901 ttttatcacc caaacctgtt agcgccttct taattctatt caatctggct tttgctctga
     3961 gactacttct tggactttca ctacttgtta gttatacgga atttgtgtaa ttagaagtga
     4021 aataatcctt tctattagta atgcgagctc gaattcgagt ctaactcctt aaccactcgg
     4081 acatagtgac ttatctgaca ctcattgtaa attaatatat atataggcat tttgtttagt
     4141 taaaggtact taagtaatta gtataaacga accaatttta taatcaggaa gttaagtgaa
     4201 tggtagcaca tgtcgtaaaa attgtgaatt tttattgaat aatattttaa atacaagcct
     4261 ttctagacta ggtatactca taaacatata tgagcaaaag gatagaggag attacattgc
     4321 atcttctaca aatttattta ttgcccttta ctgaaaaatt aaataatgag tactaaatga
     4381 taaaaagcgc tcagtacaag aaagattgca aaaatattgc attcttcatg aattaaagtt
     4441 gcatataagg catattgaaa gtaatagtac taaaacagca gttagcgaaa attaatagaa
     4501 ttatattcgc aagacaattg tgacattaaa ttaaaaaatt gtagaatttt tactatcctc
     4561 tttaacgcca tgagccttta taaaaaggtt aaattagttt taacattctt tttttgagta
     4621 agagtttaca atttatcaaa acctgtgtta ttatattcat tagtttcaat ttattagcat
     4681 ctagagaaaa atcaattggc agttacattg ttggaattta tgaagaaaaa gaatctacaa
     4741 cggagaataa gttgctgatc gctttcccta aaattgtata ttttgctgag cttattttga
     4801 catttcgttg aagttttctc taattcgcat tcattttaag taaaacaatg agaaataaaa
     4861 ttacaaaaaa tacaaattaa aatacaattt ttagctataa ttatagacga tgcccttgta
     4921 tcccattctg tctcgcttgc ccctactttt tatcttttat ataccataat gaacgctgcc
     4981 gctactaacc ataccccgat tttacatttc ggactcccaa ggacgtacaa aatagaaaac
     5041 tatagaaaaa aataatcaga aaatagcatg tcatctcttt gtaaaacgcg tttgcaagaa
     5101 gaaaggaaac aatggagaag agatcatcca tttgtatgta aattttagta aacttgaaga
     5161 aatcactaac aacttctctt acttagggat tctatgcaaa accttgtaaa tcatctgatg
     5221 gaggactcga tttaatgaat tggaaggttg gaattcactg gccgtcgttt tacaacgtcg
     5281 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     5341 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     5401 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     5461 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     5521 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     5581 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5641 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5701 ttgggtgatg gttcacgtag tgggacatcg ccctgataga cggtttttcg ccctttgacg
     5761 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     5821 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     5881 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5941 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     6001 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6061 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     6121 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     6181 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     6241 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     6301 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     6361 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     6421 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     6481 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     6541 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6601 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6661 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6721 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6781 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6841 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6901 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6961 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     7021 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     7081 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     7141 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     7201 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     7261 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     7321 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     7381 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     7441 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7501 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7561 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     7621 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     7681 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     7741 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     7801 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7861 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7921 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7981 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     8041 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     8101 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     8161 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     8221 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     8281 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     8341 agctatgacc atgattacgc caagctt
//
