LOCUS       sequence-147779-        8280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway destination vector for S. pombe. Allow N-terminal tag(s) to 
            be introduced using MCS. 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 8280)
  TITLE     Akusjarvi and Bjerling plasmids
REFERENCE   2  (bases 1 to 8280)
  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: pREP4xN_ccdB
FEATURES             Location/Qualifiers
     source          1..8280
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1228..1352
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1377..1382,1383..1400,1401..1407)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1461..2120
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1527..1546)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2462..2767
                     /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     2646..2665
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2808..2932)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     rep_origin      4037..4820
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(5170..5187)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5170..5186)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5179..5201)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5399..5854
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5486..5505)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5696..5717
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5871..5890)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5990..6012
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6050..6068)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6136..6240
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6241..6309,6310..7101)
                     /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(6459..6478)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7272..7860
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7761..7780
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8014..8031
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8148..8169
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8184..8189,8190..8207,8208..8214)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8222..8238
                     /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     8227..8249
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8246..8262
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8246..8262
                     /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 tcgagactag tcatatgaca agtttgtaca aaaaagctga acgagaaacg
     1261 taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa
     1321 tactgtaaaa cacaacatat ccagtcacta tggcggccgc attaggcacc ccaggcttta
     1381 cactttatgc ttccggctcg tataatgtgt ggattttgag ttaggatccg tcgagatttt
     1441 caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat
     1501 cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata
     1561 accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca
     1621 agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc
     1681 gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg
     1741 ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc
     1801 ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt
     1861 tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca
     1921 ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg
     1981 gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg
     2041 ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg
     2101 agtggcaggg cggggcgtaa tctagaggat ccggcttact aaaagccaga taacagtatg
     2161 cgtatttgcg cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta
     2221 tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg acagttgaca gcgacagcta
     2281 tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtaagcac aaccatgcag
     2341 aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga agggatggct
     2401 gaggtcgccc ggtttattga aatgaacggc tcttttgctg acgagaacag gggctggtga
     2461 aatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt
     2521 acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcacg
     2581 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag
     2641 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt
     2701 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg
     2761 aatataaatg tcaggctccc ttatacacag ccagtctgca ggtcgaccat agtgactgga
     2821 tatgttgtgt tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat
     2881 tgatatttat atcattttac gtttctcgtt cagctttctt gtacaaagtg gtctcgagcc
     2941 cgggcggccg ctaactaact aagggtaaaa ggaatgtctc ccttgccagt actgctaggg
     3001 tttttctttc aaactatgga agcccattca agctgcatat tacgattttg tttttcgctt
     3061 ttagaaagtg gtttagatga gataatagaa aaattcttga tctccgacaa cgagtacttt
     3121 tatttttttt gctaatcact ttactcaata ttagctcgaa atcgtagaaa cgtagacggg
     3181 tgcgggatac cgagtggtgt agttaagaat ttttataaac cacgtggccc aaaaatatga
     3241 acccaaaacg tttatacatg agtatacttt aagaaggcta taccccttcg tgttagatgt
     3301 agttttagct acccaacccg agtctatgag cttgacttca gatgtagaag gcattaaatc
     3361 gttttgaata ttaattaaaa aacgatgaaa attaaatatt taaaagcaat catacgctga
     3421 aaatttagtg ctgtggctaa tccttcaaca tggaaatgcc ataaaagtga ctttgacaaa
     3481 aaaaaaagta tatacaggta gtaaactcat ctacttcatt gactttgttt acagcatgtg
     3541 gaaggaggaa tatttattgc taaatcgtag tttaacattc aataagtaat actattgaaa
     3601 ttcgacaaga ttggccgcat ggatgaaaaa gaggcatttt gctttgggag aattagttca
     3661 aattagaact gaaaaaaaaa actttacgag gcaaaaatgt cggattgaga tcgtaaaagt
     3721 tcgctcgtcg tcttttgctt tgtgattgtt ttcatggata catcttgctg gatatttaaa
     3781 ttttagtact atgtataaga tattctataa atgttttatc acccaaacct gttagcgcct
     3841 tcttaattct attcaatctg gcttttgctc tgagactact tcttggactt tcactacttg
     3901 ttagttatac ggaatttgtg taattagaag tgaaataatc ctttctatta gtaatgcgag
     3961 ctcgaattcg agtctaactc cttaaccact cggacatagt gacttatctg acactcattg
     4021 taaattaata tatatatagg cattttgttt agttaaaggt acttaagtaa ttagtataaa
     4081 cgaaccaatt ttataatcag gaagttaagt gaatggtagc acatgtcgta aaaattgtga
     4141 atttttattg aataatattt taaatacaag cctttctaga ctaggtatac tcataaacat
     4201 atatgagcaa aaggatagag gagattacat tgcatcttct acaaatttat ttattgccct
     4261 ttactgaaaa attaaataat gagtactaaa tgataaaaag cgctcagtac aagaaagatt
     4321 gcaaaaatat tgcattcttc atgaattaaa gttgcatata aggcatattg aaagtaatag
     4381 tactaaaaca gcagttagcg aaaattaata gaattatatt cgcaagacaa ttgtgacatt
     4441 aaattaaaaa attgtagaat ttttactatc ctctttaacg ccatgagcct ttataaaaag
     4501 gttaaattag ttttaacatt ctttttttga gtaagagttt acaatttatc aaaacctgtg
     4561 ttattatatt cattagtttc aatttattag catctagaga aaaatcaatt ggcagttaca
     4621 ttgttggaat ttatgaagaa aaagaatcta caacggagaa taagttgctg atcgctttcc
     4681 ctaaaattgt atattttgct gagcttattt tgacatttcg ttgaagtttt ctctaattcg
     4741 cattcatttt aagtaaaaca atgagaaata aaattacaaa aaatacaaat taaaatacaa
     4801 tttttagcta taattataga cgatgccctt gtatcccatt ctgtctcgct tgcccctact
     4861 ttttatcttt tatataccat aatgaacgct gccgctacta accatacccc gattttacat
     4921 ttcggactcc caaggacgta caaaatagaa aactatagaa aaaaataatc agaaaatagc
     4981 atgtcatctc tttgtaaaac gcgtttgcaa gaagaaagga aacaatggag aagagatcat
     5041 ccatttgtat gtaaatttta gtaaacttga agaaatcact aacaacttct cttacttagg
     5101 gattctatgc aaaaccttgt aaatcatctg atggaggact cgatttaatg aattggaagg
     5161 ttggaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5221 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     5281 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     5341 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     5401 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     5461 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     5521 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     5581 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggaca
     5641 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     5701 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     5761 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5821 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     5881 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     5941 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     6001 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     6061 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     6121 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     6181 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     6241 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     6301 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     6361 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     6421 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     6481 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     6541 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     6601 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     6661 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6721 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6781 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6841 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6901 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6961 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     7021 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     7081 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     7141 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     7201 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     7261 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     7321 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     7381 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     7441 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     7501 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     7561 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     7621 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     7681 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7741 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7801 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7861 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7921 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     7981 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     8041 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     8101 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     8161 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     8221 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt
//
