LOCUS       sequence-147783-        8274 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Gateway destination vector for S. pombe. Allow N-terminal tag(s) to 
            be introduced using MCS. 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 8274)
  TITLE     Akusjarvi and Bjerling plasmids
REFERENCE   2  (bases 1 to 8274)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pREP82xN_ccdB
FEATURES             Location/Qualifiers
     source          1..8274
                     /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"
     protein_bind    1222..1346
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1371..1376,1377..1394,1395..1401)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1455..2114
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1521..1540)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2456..2761
                     /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     2640..2659
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2802..2926)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     rep_origin      4031..4814
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(5164..5181)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5164..5180)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5173..5195)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5393..5848
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5480..5499)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5690..5711
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5865..5884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5984..6006
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6044..6062)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6130..6234
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6235..6303,6304..7095)
                     /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(6453..6472)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7266..7854
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7755..7774
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8008..8025
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8142..8163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8178..8183,8184..8201,8202..8208)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8216..8232
                     /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     8221..8243
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8240..8256
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8240..8256
                     /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 tacctcgaga ctagtcatat gacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat
     1261 gatataaata tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt
     1321 aaaacacaac atatccagtc actatggcgg ccgcattagg caccccaggc tttacacttt
     1381 atgcttccgg ctcgtataat gtgtggattt tgagttagga tccgtcgaga ttttcaggag
     1441 ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat
     1501 ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga
     1561 ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt
     1621 atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg
     1681 caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc
     1741 atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt
     1801 ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta
     1861 aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt
     1921 ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat
     1981 attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt
     2041 gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc
     2101 agggcggggc gtaatctaga ggatccggct tactaaaagc cagataacag tatgcgtatt
     2161 tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa
     2221 aaagaggtat gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt
     2281 gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat gcagaatgaa
     2341 gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat ggctgaggtc
     2401 gcccggttta ttgaaatgaa cggctctttt gctgacgaga acaggggctg gtgaaatgca
     2461 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
     2521 tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
     2581 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
     2641 catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga
     2701 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
     2761 aatgtcaggc tcccttatac acagccagtc tgcaggtcga ccatagtgac tggatatgtt
     2821 gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat
     2881 ttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggtctcg agcccgggcg
     2941 gccgctaact aactaagggt aaaaggaatg tctcccttgc cagtactgct agggtttttc
     3001 tttcaaacta tggaagccca ttcaagctgc atattacgat tttgtttttc gcttttagaa
     3061 agtggtttag atgagataat agaaaaattc ttgatctccg acaacgagta cttttatttt
     3121 ttttgctaat cactttactc aatattagct cgaaatcgta gaaacgtaga cgggtgcggg
     3181 ataccgagtg gtgtagttaa gaatttttat aaaccacgtg gcccaaaaat atgaacccaa
     3241 aacgtttata catgagtata ctttaagaag gctatacccc ttcgtgttag atgtagtttt
     3301 agctacccaa cccgagtcta tgagcttgac ttcagatgta gaaggcatta aatcgttttg
     3361 aatattaatt aaaaaacgat gaaaattaaa tatttaaaag caatcatacg ctgaaaattt
     3421 agtgctgtgg ctaatccttc aacatggaaa tgccataaaa gtgactttga caaaaaaaaa
     3481 agtatataca ggtagtaaac tcatctactt cattgacttt gtttacagca tgtggaagga
     3541 ggaatattta ttgctaaatc gtagtttaac attcaataag taatactatt gaaattcgac
     3601 aagattggcc gcatggatga aaaagaggca ttttgctttg ggagaattag ttcaaattag
     3661 aactgaaaaa aaaaacttta cgaggcaaaa atgtcggatt gagatcgtaa aagttcgctc
     3721 gtcgtctttt gctttgtgat tgttttcatg gatacatctt gctggatatt taaattttag
     3781 tactatgtat aagatattct ataaatgttt tatcacccaa acctgttagc gccttcttaa
     3841 ttctattcaa tctggctttt gctctgagac tacttcttgg actttcacta cttgttagtt
     3901 atacggaatt tgtgtaatta gaagtgaaat aatcctttct attagtaatg cgagctcgaa
     3961 ttcgagtcta actccttaac cactcggaca tagtgactta tctgacactc attgtaaatt
     4021 aatatatata taggcatttt gtttagttaa aggtacttaa gtaattagta taaacgaacc
     4081 aattttataa tcaggaagtt aagtgaatgg tagcacatgt cgtaaaaatt gtgaattttt
     4141 attgaataat attttaaata caagcctttc tagactaggt atactcataa acatatatga
     4201 gcaaaaggat agaggagatt acattgcatc ttctacaaat ttatttattg ccctttactg
     4261 aaaaattaaa taatgagtac taaatgataa aaagcgctca gtacaagaaa gattgcaaaa
     4321 atattgcatt cttcatgaat taaagttgca tataaggcat attgaaagta atagtactaa
     4381 aacagcagtt agcgaaaatt aatagaatta tattcgcaag acaattgtga cattaaatta
     4441 aaaaattgta gaatttttac tatcctcttt aacgccatga gcctttataa aaaggttaaa
     4501 ttagttttaa cattcttttt ttgagtaaga gtttacaatt tatcaaaacc tgtgttatta
     4561 tattcattag tttcaattta ttagcatcta gagaaaaatc aattggcagt tacattgttg
     4621 gaatttatga agaaaaagaa tctacaacgg agaataagtt gctgatcgct ttccctaaaa
     4681 ttgtatattt tgctgagctt attttgacat ttcgttgaag ttttctctaa ttcgcattca
     4741 ttttaagtaa aacaatgaga aataaaatta caaaaaatac aaattaaaat acaattttta
     4801 gctataatta tagacgatgc ccttgtatcc cattctgtct cgcttgcccc tactttttat
     4861 cttttatata ccataatgaa cgctgccgct actaaccata ccccgatttt acatttcgga
     4921 ctcccaagga cgtacaaaat agaaaactat agaaaaaaat aatcagaaaa tagcatgtca
     4981 tctctttgta aaacgcgttt gcaagaagaa aggaaacaat ggagaagaga tcatccattt
     5041 gtatgtaaat tttagtaaac ttgaagaaat cactaacaac ttctcttact tagggattct
     5101 atgcaaaacc ttgtaaatca tctgatggag gactcgattt aatgaattgg aaggttggaa
     5161 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     5221 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     5281 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     5341 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     5401 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5461 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5521 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5581 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gacatcgccc
     5641 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5701 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     5761 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5821 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     5881 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5941 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     6001 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     6061 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     6121 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6181 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6241 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6301 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6361 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6421 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6481 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6541 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6601 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6661 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6721 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6781 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6841 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6901 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6961 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     7021 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     7081 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     7141 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     7201 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     7261 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7321 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7381 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     7441 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7501 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7561 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7621 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7681 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7741 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7801 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7861 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7921 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7981 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     8041 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     8101 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     8161 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     8221 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctt
//
