LOCUS       sequence-147775-        8370 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* (ATAAA) promoter, ura4+ 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8370)
  TITLE     Akusjarvi and Bjerling plasmids
REFERENCE   2  (bases 1 to 8370)
  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: pREP42xV5_ccdB
FEATURES             Location/Qualifiers
     source          1..8370
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        49..1203
                     /label=nmt1 P41 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring medium strength thiamine-repressible expression"
     CDS             1219..1260
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1267..1308
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    1318..1442
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1467..1472,1473..1490,1491..1497)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1551..2210
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1617..1636)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2552..2857
                     /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     2736..2755
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2898..3022)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     rep_origin      4127..4910
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(5260..5277)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5260..5276)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5269..5291)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5489..5944
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5576..5595)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5786..5807
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5961..5980)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6080..6102
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6140..6158)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6226..6330
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6331..6399,6400..7191)
                     /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(6549..6568)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7362..7950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7851..7870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8104..8121
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8238..8259
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8274..8279,8280..8297,8298..8304)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8312..8328
                     /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     8317..8339
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8336..8352
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8336..8352
                     /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 agataaagga agaggaatcc tggcatatca tcaattgaat
     1141 aagttgaatt aattatttca atctcattct cactttctga cttatagtcg ctttgttaaa
     1201 tcatacctcg agtctatggg taaacctatt cctaaccctt tgttaggctt ggacagtact
     1261 ggtgctggta agcctattcc caatcccttg cttggtcttg actctactgg tcatatgaca
     1321 agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa tatattaaat
     1381 tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta
     1441 tggcggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt
     1501 ggattttgag ttaggatccg tcgagatttt caggagctaa ggaagctaaa atggagaaaa
     1561 aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg
     1621 catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct
     1681 ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg
     1741 cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga
     1801 tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat
     1861 cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg
     1921 tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt
     1981 tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg
     2041 acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc
     2101 tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa
     2161 tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa tctagaggat
     2221 ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa
     2281 gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg
     2341 tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat
     2401 atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc
     2461 tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc
     2521 tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag
     2581 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg
     2641 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     2701 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     2761 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     2821 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag
     2881 ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct
     2941 gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt
     3001 cagctttctt gtacaaagtg gtctcgagcc cgggcggccg ctaactaact aagggtaaaa
     3061 ggaatgtctc ccttgccagt actgctaggg tttttctttc aaactatgga agcccattca
     3121 agctgcatat tacgattttg tttttcgctt ttagaaagtg gtttagatga gataatagaa
     3181 aaattcttga tctccgacaa cgagtacttt tatttttttt gctaatcact ttactcaata
     3241 ttagctcgaa atcgtagaaa cgtagacggg tgcgggatac cgagtggtgt agttaagaat
     3301 ttttataaac cacgtggccc aaaaatatga acccaaaacg tttatacatg agtatacttt
     3361 aagaaggcta taccccttcg tgttagatgt agttttagct acccaacccg agtctatgag
     3421 cttgacttca gatgtagaag gcattaaatc gttttgaata ttaattaaaa aacgatgaaa
     3481 attaaatatt taaaagcaat catacgctga aaatttagtg ctgtggctaa tccttcaaca
     3541 tggaaatgcc ataaaagtga ctttgacaaa aaaaaaagta tatacaggta gtaaactcat
     3601 ctacttcatt gactttgttt acagcatgtg gaaggaggaa tatttattgc taaatcgtag
     3661 tttaacattc aataagtaat actattgaaa ttcgacaaga ttggccgcat ggatgaaaaa
     3721 gaggcatttt gctttgggag aattagttca aattagaact gaaaaaaaaa actttacgag
     3781 gcaaaaatgt cggattgaga tcgtaaaagt tcgctcgtcg tcttttgctt tgtgattgtt
     3841 ttcatggata catcttgctg gatatttaaa ttttagtact atgtataaga tattctataa
     3901 atgttttatc acccaaacct gttagcgcct tcttaattct attcaatctg gcttttgctc
     3961 tgagactact tcttggactt tcactacttg ttagttatac ggaatttgtg taattagaag
     4021 tgaaataatc ctttctatta gtaatgcgag ctcgaattcg agtctaactc cttaaccact
     4081 cggacatagt gacttatctg acactcattg taaattaata tatatatagg cattttgttt
     4141 agttaaaggt acttaagtaa ttagtataaa cgaaccaatt ttataatcag gaagttaagt
     4201 gaatggtagc acatgtcgta aaaattgtga atttttattg aataatattt taaatacaag
     4261 cctttctaga ctaggtatac tcataaacat atatgagcaa aaggatagag gagattacat
     4321 tgcatcttct acaaatttat ttattgccct ttactgaaaa attaaataat gagtactaaa
     4381 tgataaaaag cgctcagtac aagaaagatt gcaaaaatat tgcattcttc atgaattaaa
     4441 gttgcatata aggcatattg aaagtaatag tactaaaaca gcagttagcg aaaattaata
     4501 gaattatatt cgcaagacaa ttgtgacatt aaattaaaaa attgtagaat ttttactatc
     4561 ctctttaacg ccatgagcct ttataaaaag gttaaattag ttttaacatt ctttttttga
     4621 gtaagagttt acaatttatc aaaacctgtg ttattatatt cattagtttc aatttattag
     4681 catctagaga aaaatcaatt ggcagttaca ttgttggaat ttatgaagaa aaagaatcta
     4741 caacggagaa taagttgctg atcgctttcc ctaaaattgt atattttgct gagcttattt
     4801 tgacatttcg ttgaagtttt ctctaattcg cattcatttt aagtaaaaca atgagaaata
     4861 aaattacaaa aaatacaaat taaaatacaa tttttagcta taattataga cgatgccctt
     4921 gtatcccatt ctgtctcgct tgcccctact ttttatcttt tatataccat aatgaacgct
     4981 gccgctacta accatacccc gattttacat ttcggactcc caaggacgta caaaatagaa
     5041 aactatagaa aaaaataatc agaaaatagc atgtcatctc tttgtaaaac gcgtttgcaa
     5101 gaagaaagga aacaatggag aagagatcat ccatttgtat gtaaatttta gtaaacttga
     5161 agaaatcact aacaacttct cttacttagg gattctatgc aaaaccttgt aaatcatctg
     5221 atggaggact cgatttaatg aattggaagg ttggaattca ctggccgtcg ttttacaacg
     5281 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     5341 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     5401 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5461 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     5521 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     5581 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5641 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5701 gatttgggtg atggttcacg tagtgggaca tcgccctgat agacggtttt tcgccctttg
     5761 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5821 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     5881 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     5941 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     6001 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6061 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6121 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     6181 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6241 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6301 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6361 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6421 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6481 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6541 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6601 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6661 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6721 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6781 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6841 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6901 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6961 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     7021 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     7081 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     7141 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7201 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7261 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7501 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7741 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7801 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7861 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7921 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7981 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     8041 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     8101 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     8161 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     8221 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     8281 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     8341 aacagctatg accatgatta cgccaagctt
//
