LOCUS       sequence-147781-        8277 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* 
            (ATAAA) promoter, ura4+ selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8277)
  TITLE     Akusjarvi and Bjerling plasmids
REFERENCE   2  (bases 1 to 8277)
  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: pREP42xN_ccdB
FEATURES             Location/Qualifiers
     source          1..8277
                     /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"
     protein_bind    1225..1349
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1374..1379,1380..1397,1398..1404)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1458..2117
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1524..1543)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2459..2764
                     /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     2643..2662
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2805..2929)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     rep_origin      4034..4817
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(5167..5184)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5167..5183)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5176..5198)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5396..5851
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5483..5502)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5693..5714
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5868..5887)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5987..6009
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6047..6065)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6133..6237
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6238..6306,6307..7098)
                     /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(6456..6475)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7269..7857
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7758..7777
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8011..8028
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8145..8166
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8181..8186,8187..8204,8205..8211)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8219..8235
                     /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     8224..8246
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8243..8259
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8243..8259
                     /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 agactagtca tatgacaagt ttgtacaaaa aagctgaacg agaaacgtaa
     1261 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
     1321 tgtaaaacac aacatatcca gtcactatgg cggccgcatt aggcacccca ggctttacac
     1381 tttatgcttc cggctcgtat aatgtgtgga ttttgagtta ggatccgtcg agattttcag
     1441 gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc
     1501 aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc
     1561 agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt
     1621 tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta
     1681 tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt
     1741 tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc
     1801 agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc
     1861 ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca
     1921 gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca
     1981 aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg
     2041 tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt
     2101 ggcagggcgg ggcgtaatct agaggatccg gcttactaaa agccagataa cagtatgcgt
     2161 atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt
     2221 caaaaagagg tatgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca
     2281 gttgctcaag gcatatatga tgtcaatatc tccggtctgg taagcacaac catgcagaat
     2341 gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag
     2401 gtcgcccggt ttattgaaat gaacggctct tttgctgacg agaacagggg ctggtgaaat
     2461 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
     2521 gagtgatatt attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct
     2581 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
     2641 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
     2701 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     2761 ataaatgtca ggctccctta tacacagcca gtctgcaggt cgaccatagt gactggatat
     2821 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
     2881 tatttatatc attttacgtt tctcgttcag ctttcttgta caaagtggtc tcgagcccgg
     2941 gcggccgcta actaactaag ggtaaaagga atgtctccct tgccagtact gctagggttt
     3001 ttctttcaaa ctatggaagc ccattcaagc tgcatattac gattttgttt ttcgctttta
     3061 gaaagtggtt tagatgagat aatagaaaaa ttcttgatct ccgacaacga gtacttttat
     3121 tttttttgct aatcacttta ctcaatatta gctcgaaatc gtagaaacgt agacgggtgc
     3181 gggataccga gtggtgtagt taagaatttt tataaaccac gtggcccaaa aatatgaacc
     3241 caaaacgttt atacatgagt atactttaag aaggctatac cccttcgtgt tagatgtagt
     3301 tttagctacc caacccgagt ctatgagctt gacttcagat gtagaaggca ttaaatcgtt
     3361 ttgaatatta attaaaaaac gatgaaaatt aaatatttaa aagcaatcat acgctgaaaa
     3421 tttagtgctg tggctaatcc ttcaacatgg aaatgccata aaagtgactt tgacaaaaaa
     3481 aaaagtatat acaggtagta aactcatcta cttcattgac tttgtttaca gcatgtggaa
     3541 ggaggaatat ttattgctaa atcgtagttt aacattcaat aagtaatact attgaaattc
     3601 gacaagattg gccgcatgga tgaaaaagag gcattttgct ttgggagaat tagttcaaat
     3661 tagaactgaa aaaaaaaact ttacgaggca aaaatgtcgg attgagatcg taaaagttcg
     3721 ctcgtcgtct tttgctttgt gattgttttc atggatacat cttgctggat atttaaattt
     3781 tagtactatg tataagatat tctataaatg ttttatcacc caaacctgtt agcgccttct
     3841 taattctatt caatctggct tttgctctga gactacttct tggactttca ctacttgtta
     3901 gttatacgga atttgtgtaa ttagaagtga aataatcctt tctattagta atgcgagctc
     3961 gaattcgagt ctaactcctt aaccactcgg acatagtgac ttatctgaca ctcattgtaa
     4021 attaatatat atataggcat tttgtttagt taaaggtact taagtaatta gtataaacga
     4081 accaatttta taatcaggaa gttaagtgaa tggtagcaca tgtcgtaaaa attgtgaatt
     4141 tttattgaat aatattttaa atacaagcct ttctagacta ggtatactca taaacatata
     4201 tgagcaaaag gatagaggag attacattgc atcttctaca aatttattta ttgcccttta
     4261 ctgaaaaatt aaataatgag tactaaatga taaaaagcgc tcagtacaag aaagattgca
     4321 aaaatattgc attcttcatg aattaaagtt gcatataagg catattgaaa gtaatagtac
     4381 taaaacagca gttagcgaaa attaatagaa ttatattcgc aagacaattg tgacattaaa
     4441 ttaaaaaatt gtagaatttt tactatcctc tttaacgcca tgagccttta taaaaaggtt
     4501 aaattagttt taacattctt tttttgagta agagtttaca atttatcaaa acctgtgtta
     4561 ttatattcat tagtttcaat ttattagcat ctagagaaaa atcaattggc agttacattg
     4621 ttggaattta tgaagaaaaa gaatctacaa cggagaataa gttgctgatc gctttcccta
     4681 aaattgtata ttttgctgag cttattttga catttcgttg aagttttctc taattcgcat
     4741 tcattttaag taaaacaatg agaaataaaa ttacaaaaaa tacaaattaa aatacaattt
     4801 ttagctataa ttatagacga tgcccttgta tcccattctg tctcgcttgc ccctactttt
     4861 tatcttttat ataccataat gaacgctgcc gctactaacc ataccccgat tttacatttc
     4921 ggactcccaa ggacgtacaa aatagaaaac tatagaaaaa aataatcaga aaatagcatg
     4981 tcatctcttt gtaaaacgcg tttgcaagaa gaaaggaaac aatggagaag agatcatcca
     5041 tttgtatgta aattttagta aacttgaaga aatcactaac aacttctctt acttagggat
     5101 tctatgcaaa accttgtaaa tcatctgatg gaggactcga tttaatgaat tggaaggttg
     5161 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     5221 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     5281 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     5341 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     5401 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     5461 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     5521 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     5581 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggacatcg
     5641 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5701 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     5761 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5821 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     5881 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5941 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     6001 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     6061 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     6121 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     6181 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     6241 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     6301 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     6361 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     6421 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     6481 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     6541 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     6601 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     6661 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6721 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6781 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6841 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6901 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6961 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     7021 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     7081 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     7141 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     7201 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     7261 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     7321 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     7381 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     7441 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7501 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7561 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7621 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7681 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7741 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7801 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7861 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7921 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7981 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     8041 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     8101 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     8161 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     8221 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagctt
//
