LOCUS       sequence-147773-        8373 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 (ATATATAAA) promoter, ura4+ 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8373)
  TITLE     Akusjarvi and Bjerling plasmids
REFERENCE   2  (bases 1 to 8373)
  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: pREP4xV5_ccdB
FEATURES             Location/Qualifiers
     source          1..8373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1222..1263
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1270..1311
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    1321..1445
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1470..1475,1476..1493,1494..1500)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1554..2213
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1620..1639)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2555..2860
                     /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     2739..2758
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2901..3025)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     rep_origin      4130..4913
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(5263..5280)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5263..5279)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5272..5294)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5492..5947
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5579..5598)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5789..5810
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5964..5983)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6083..6105
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6143..6161)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6229..6333
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6334..6402,6403..7194)
                     /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(6552..6571)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7365..7953
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7854..7873
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8107..8124
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8241..8262
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8277..8282,8283..8300,8301..8307)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8315..8331
                     /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     8320..8342
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8339..8355
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8339..8355
                     /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 agatatataa aggaagagga atcctggcat atcatcaatt
     1141 gaataagttg aattaattat ttcaatctca ttctcacttt ctgacttata gtcgctttgt
     1201 taaattggcc tcgagtctat gggtaaacct attcctaacc ctttgttagg cttggacagt
     1261 actggtgctg gtaagcctat tcccaatccc ttgcttggtc ttgactctac tggtcatatg
     1321 acaagtttgt acaaaaaagc tgaacgagaa acgtaaaatg atataaatat caatatatta
     1381 aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca
     1441 ctatggcggc cgcattaggc accccaggct ttacacttta tgcttccggc tcgtataatg
     1501 tgtggatttt gagttaggat ccgtcgagat tttcaggagc taaggaagct aaaatggaga
     1561 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
     1621 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
     1681 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
     1741 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
     1801 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
     1861 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
     1921 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
     1981 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
     2041 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     2101 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     2161 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg taatctagag
     2221 gatccggctt actaaaagcc agataacagt atgcgtattt gcgcgctgat ttttgcggta
     2281 taagaatata tactgatatg tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca
     2341 gcgtattaca gtgacagttg acagcgacag ctatcagttg ctcaaggcat atatgatgtc
     2401 aatatctccg gtctggtaag cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa
     2461 cgctggaaag cggaaaatca ggaagggatg gctgaggtcg cccggtttat tgaaatgaac
     2521 ggctcttttg ctgacgagaa caggggctgg tgaaatgcag tttaaggttt acacctataa
     2581 aagagagagc cgttatcgtc tgtttgtgga tgtacagagt gatattattg acacgcccgg
     2641 gcgacggatg gtgatccccc tggccagtgc acgtctgctg tcagataaag tctcccgtga
     2701 actttacccg gtggtgcata tcggggatga aagctggcgc atgatgacca ccgatatggc
     2761 cagtgtgccg gtctccgtta tcggggaaga agtggctgat ctcagccacc gcgaaaatga
     2821 catcaaaaac gccattaacc tgatgttctg gggaatataa atgtcaggct cccttataca
     2881 cagccagtct gcaggtcgac catagtgact ggatatgttg tgttttacag tattatgtag
     2941 tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc
     3001 gttcagcttt cttgtacaaa gtggtctcga gcccgggcgg ccgctaacta actaagggta
     3061 aaaggaatgt ctcccttgcc agtactgcta gggtttttct ttcaaactat ggaagcccat
     3121 tcaagctgca tattacgatt ttgtttttcg cttttagaaa gtggtttaga tgagataata
     3181 gaaaaattct tgatctccga caacgagtac ttttattttt tttgctaatc actttactca
     3241 atattagctc gaaatcgtag aaacgtagac gggtgcggga taccgagtgg tgtagttaag
     3301 aatttttata aaccacgtgg cccaaaaata tgaacccaaa acgtttatac atgagtatac
     3361 tttaagaagg ctatacccct tcgtgttaga tgtagtttta gctacccaac ccgagtctat
     3421 gagcttgact tcagatgtag aaggcattaa atcgttttga atattaatta aaaaacgatg
     3481 aaaattaaat atttaaaagc aatcatacgc tgaaaattta gtgctgtggc taatccttca
     3541 acatggaaat gccataaaag tgactttgac aaaaaaaaaa gtatatacag gtagtaaact
     3601 catctacttc attgactttg tttacagcat gtggaaggag gaatatttat tgctaaatcg
     3661 tagtttaaca ttcaataagt aatactattg aaattcgaca agattggccg catggatgaa
     3721 aaagaggcat tttgctttgg gagaattagt tcaaattaga actgaaaaaa aaaactttac
     3781 gaggcaaaaa tgtcggattg agatcgtaaa agttcgctcg tcgtcttttg ctttgtgatt
     3841 gttttcatgg atacatcttg ctggatattt aaattttagt actatgtata agatattcta
     3901 taaatgtttt atcacccaaa cctgttagcg ccttcttaat tctattcaat ctggcttttg
     3961 ctctgagact acttcttgga ctttcactac ttgttagtta tacggaattt gtgtaattag
     4021 aagtgaaata atcctttcta ttagtaatgc gagctcgaat tcgagtctaa ctccttaacc
     4081 actcggacat agtgacttat ctgacactca ttgtaaatta atatatatat aggcattttg
     4141 tttagttaaa ggtacttaag taattagtat aaacgaacca attttataat caggaagtta
     4201 agtgaatggt agcacatgtc gtaaaaattg tgaattttta ttgaataata ttttaaatac
     4261 aagcctttct agactaggta tactcataaa catatatgag caaaaggata gaggagatta
     4321 cattgcatct tctacaaatt tatttattgc cctttactga aaaattaaat aatgagtact
     4381 aaatgataaa aagcgctcag tacaagaaag attgcaaaaa tattgcattc ttcatgaatt
     4441 aaagttgcat ataaggcata ttgaaagtaa tagtactaaa acagcagtta gcgaaaatta
     4501 atagaattat attcgcaaga caattgtgac attaaattaa aaaattgtag aatttttact
     4561 atcctcttta acgccatgag cctttataaa aaggttaaat tagttttaac attctttttt
     4621 tgagtaagag tttacaattt atcaaaacct gtgttattat attcattagt ttcaatttat
     4681 tagcatctag agaaaaatca attggcagtt acattgttgg aatttatgaa gaaaaagaat
     4741 ctacaacgga gaataagttg ctgatcgctt tccctaaaat tgtatatttt gctgagctta
     4801 ttttgacatt tcgttgaagt tttctctaat tcgcattcat tttaagtaaa acaatgagaa
     4861 ataaaattac aaaaaataca aattaaaata caatttttag ctataattat agacgatgcc
     4921 cttgtatccc attctgtctc gcttgcccct actttttatc ttttatatac cataatgaac
     4981 gctgccgcta ctaaccatac cccgatttta catttcggac tcccaaggac gtacaaaata
     5041 gaaaactata gaaaaaaata atcagaaaat agcatgtcat ctctttgtaa aacgcgtttg
     5101 caagaagaaa ggaaacaatg gagaagagat catccatttg tatgtaaatt ttagtaaact
     5161 tgaagaaatc actaacaact tctcttactt agggattcta tgcaaaacct tgtaaatcat
     5221 ctgatggagg actcgattta atgaattgga aggttggaat tcactggccg tcgttttaca
     5281 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     5341 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     5401 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5461 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     5521 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     5581 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5641 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5701 cttgatttgg gtgatggttc acgtagtggg acatcgccct gatagacggt ttttcgccct
     5761 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5821 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     5881 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     5941 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     6001 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     6061 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     6121 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     6181 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     6241 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6301 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6361 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6421 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6481 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6541 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6601 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6661 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6721 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6781 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6841 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6901 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6961 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     7021 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     7081 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     7141 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     7201 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     7261 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7321 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7381 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7441 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7501 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7561 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7621 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7681 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7741 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7801 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7861 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7921 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7981 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     8041 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     8101 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     8161 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     8221 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     8281 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     8341 ggaaacagct atgaccatga ttacgccaag ctt
//
