LOCUS       sequence-82000-e        8795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Same as pREP41, with better MCS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8795)
  TITLE     various
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8795)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pREP41-MCS+
FEATURES             Location/Qualifiers
     source          1..8795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(722..1816)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1817..2224)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     promoter        2514..3674
                     /label=nmt1 P41 promoter
                     /note="mutant nmt1 promoter from Schizosaccharomyces pombe,
                     conferring medium strength thiamine-repressible expression"
     rep_origin      4791..5574
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(5924..5941)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5924..5940)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5933..5955)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6153..6608
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6240..6259)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6450..6471
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6625..6644)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6744..6766
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6804..6822)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6890..6994
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6995..7063,7064..7855)
                     /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(7213..7232)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8026..8614
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8515..8534
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8768..8785
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 tcgatcgact acgtcgttaa ggccgtttct gacagagtaa aattcttgag ggaactttca
      301 ccattatggg aaatggttca agaaggtatt gacttaaact ccatcaaatg gtcaggtcat
      361 tgagtgtttt ttatttgttg tatttttttt tttttagaga aaatcctcca atatataaat
      421 taggaatcat agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
      481 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
      541 aatttgctta cctgtattcc tttacatcct cctttttctc cttcttgata aatgtatgta
      601 gattgcgtat atagtttcgt ctaccctatg aacatattcc attttgtaat ttcgtgtcgt
      661 ttctattatg aatttcattt ataaagttta tgtacaaata tcataaaaaa agagaatctt
      721 tttaagcaag gattttctta acttcttcgg cgacagcatc accgacttcc gtggtactgt
      781 tggaaccacc taaatcacca gttctgatac ctgcatccaa aaccttttta actgcatctt
      841 caatggcctt accttcttca ggcaagttca atgacaattt caacatcatt gcagcagaca
      901 agatagtggc gatagggttg accttattct ttggcaaatc tggagcagaa ccgtggcatg
      961 gttcgtacaa accaaatgcg gtgttcttgt ctggcaaaga ggccaaggac gcagatggca
     1021 acaaacccaa ggaacctggg ataacggagg cttcatcgga gatgatatca ccaaacatgt
     1081 tgctggtgat tataatacca tttaggtggg ttgggttctt aactaggatc atggcggcag
     1141 aatcaatcaa ttgatgttga accttcaatg tagggaattc gttcttgatg gtttcctcca
     1201 cagtttttct ccataatctt gaagaggcca aaacattagc tttatccaag gaccaaatag
     1261 gcaatggtgg ctcatgttgt agggccatga aagcggccat tcttgtgatt ctttgcactt
     1321 ctggaacggt gtattgttca ctatcccaag cgacaccatc accatcgtct tcctttctct
     1381 taccaaagta aatacctccc actaattctc tgacaacaac gaagtcagta cctttagcaa
     1441 attgtggctt gattggagat aagtctaaaa gagagtcgga tgcaaagtta catggtctta
     1501 agttggcgta caattgaagt tctttacgga tttttagtaa accttgttca ggtctaacac
     1561 taccggtacc ccatttagga ccacccacag cacctaacaa aacggcatca accttcttgg
     1621 aggcttccag cgcctcatct ggaagtggga cacctgtagc atcgatagca gcaccaccaa
     1681 ttaaatgatt ttcgaaatcg aacttgacat tggaacgaac atcagaaata gctttaagaa
     1741 ccttaatggc ttcggctgtg atttcttgac caacgtggtc acctggcaaa acgacgatct
     1801 tcttaggggc agacattaga atggtatatc cttgaaatat atatatatat attgctgaaa
     1861 tgtaaaaggt aagaaaagtt agaaagtaag acgattgcta accacctatt ggaaaaaaca
     1921 ataggtcctt aaataatatt gtcaacttca agtattgtga tgcaagcatt tagtcatgaa
     1981 cgcttctcta ttctatatga aaagccggtt ccgcggctct cacctttcct ttttctccca
     2041 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc
     2101 agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa
     2161 aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga gtattcccac
     2221 agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg gccaaacaac
     2281 caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt ttgaacacac
     2341 atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg atgtaattgt
     2401 tgggattcca tttttaataa ggcaataata ttaggtatgt agatatacta gaagttctcc
     2461 tcgacaagct tgcatgcctg caggtcgatc gactctagag gatcagaaaa ttatcgccat
     2521 aaaagacaga ataagtcatc agcggttgtt tcatttccta tatttttttt ttattttttt
     2581 attttttaat aagggaaaat ttaacgtcta aggatacaga agattgttag cacattaaag
     2641 taataaaggc ttaagtagta agtgccttag catgttattg tatttcaaag gacataatct
     2701 aaaataataa caatatcatt tctcacaagt tattcaattt tctttttttt ttctaataat
     2761 atcaagaatg tattatttgt ttgacataag tcaactaatt tatttaatat gctggattaa
     2821 tcttgcagac atgtaaatta acaagtttta gtcaaataac gttgaagttt caatgaactc
     2881 aaataatttc tctttttttt tatataacca taagtctaat ctgatttata ttttccgcag
     2941 ggatcaactg aagttatgac atttggattg gatcacttat aaccttggtc gccaaataat
     3001 acaaaaatca gcgttataaa acaaagaagg tttttgttaa gaaattaatc ctctttcttg
     3061 ataagaaagt tgaaccgaaa ttgcagatac tgatatatga aaataatacc cacaattttg
     3121 ggaatagcgc aagcctcaat ttaaacaata ggtgaggaca catgataatg acctcaatga
     3181 ttgttagaag aaaagagcct cattacaaaa tcgaaaaatg aatggttggg tacaagtttc
     3241 caaaacatgg taaagtggac tttgcgtatg agacgtaaat agaaaaaaac acttgttata
     3301 tgttttctag aattattgtt gtctctttat ggttggatga tgcaaaatag taatttcggt
     3361 tagttgctgt aaaacaccac gagacaaata gatatggata tttattaaat caggaaaaac
     3421 gtaactctcg gctactggat ggttcagtca cccaacgatt actggggaga gaaaacaggg
     3481 caaaagcaaa gcttaaagga atccgattgt cattcggcaa tgtgcagcga aactaaaaac
     3541 cggataatgg acctgttaat cgaaacattg aagataaagg aagaggaatc ctggcatatc
     3601 atcaattgaa taagttgaat taattatttc aatctcattc tcactttctg acttatagtc
     3661 gctttgttaa atcatatgct cgagagatct gtcgacccat gggctagcgg atccccgggt
     3721 aaaaggaatg tctcccttgc cagtactgct agggtttttc tttcaaacta tggaagccca
     3781 ttcaagctgc atattacgat tttgtttttc gcttttagaa agtggtttag atgagataat
     3841 agaaaaattc ttgatctccg acaacgagta cttttatttt ttttgctaat cactttactc
     3901 aatattagct cgaaatcgta gaaacgtaga cgggtgcggg ataccgagtg gtgtagttaa
     3961 gaatttttat aaaccacgtg gcccaaaaat atgaacccaa aacgtttata catgagtata
     4021 ctttaagaag gctatacccc ttcgtgttag atgtagtttt agctacccaa cccgagtcta
     4081 tgagcttgac ttcagatgta gaaggcatta aatcgttttg aatattaatt aaaaaacgat
     4141 gaaaattaaa tatttaaaag caatcatacg ctgaaaattt agtgctgtgg ctaatccttc
     4201 aacatggaaa tgccataaaa gtgactttga caaaaaaaaa agtatataca ggtagtaaac
     4261 tcatctactt cattgacttt gtttacagca tgtggaagga ggaatattta ttgctaaatc
     4321 gtagtttaac attcaataag taatactatt gaaattcgac aagattggcc gcatggatga
     4381 aaaagaggca ttttgctttg ggagaattag ttcaaattag aactgaaaaa aaaaacttta
     4441 cgaggcaaaa atgtcggatt gagatcgtaa aagttcgctc gtcgtctttt gctttgtgat
     4501 tgttttcatg gatacatctt gctggatatt taaattttag tactatgtat aagatattct
     4561 ataaatgttt tatcacccaa acctgttagc gccttcttaa ttctattcaa tctggctttt
     4621 gctctgagac tacttcttgg actttcacta cttgttagtt atacggaatt tgtgtaatta
     4681 gaagtgaaat aatcctttct attagtaatg cgagctcgaa ttcgagtcta actccttaac
     4741 cactcggaca tagtgactta tctgacactc attgtaaatt aatatatata taggcatttt
     4801 gtttagttaa aggtacttaa gtaattagta taaacgaacc aattttataa tcaggaagtt
     4861 aagtgaatgg tagcacatgt cgtaaaaatt gtgaattttt attgaataat attttaaata
     4921 caagcctttc tagactaggt atactcataa acatatatga gcaaaaggat agaggagatt
     4981 acattgcatc ttctacaaat ttatttattg ccctttactg aaaaattaaa taatgagtac
     5041 taaatgataa aaagcgctca gtacaagaaa gattgcaaaa atattgcatt cttcatgaat
     5101 taaagttgca tataaggcat attgaaagta atagtactaa aacagcagtt agcgaaaatt
     5161 aatagaatta tattcgcaag acaattgtga cattaaatta aaaaattgta gaatttttac
     5221 tatcctcttt aacgccatga gcctttataa aaaggttaaa ttagttttaa cattcttttt
     5281 ttgagtaaga gtttacaatt tatcaaaacc tgtgttatta tattcattag tttcaattta
     5341 ttagcatcta gagaaaaatc aattggcagt tacattgttg gaatttatga agaaaaagaa
     5401 tctacaacgg agaataagtt gctgatcgct ttccctaaaa ttgtatattt tgctgagctt
     5461 attttgacat ttcgttgaag ttttctctaa ttcgcattca ttttaagtaa aacaatgaga
     5521 aataaaatta caaaaaatac aaattaaaat acaattttta gctataatta tagacgatgc
     5581 ccttgtatcc cattctgtct cgcttgcccc tactttttat cttttatata ccataatgaa
     5641 cgctgccgct actaaccata ccccgatttt acatttcgga ctcccaagga cgtacaaaat
     5701 agaaaactat agaaaaaaat aatcagaaaa tagcatgtca tctctttgta aaacgcgttt
     5761 gcaagaagaa aggaaacaat ggagaagaga tcatccattt gtatgtaaat tttagtaaac
     5821 ttgaagaaat cactaacaac ttctcttact tagggattct atgcaaaacc ttgtaaatca
     5881 tctgatggag gactcgattt aatgaattgg aaggttggaa ttcactggcc gtcgttttac
     5941 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
     6001 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
     6061 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     6121 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     6181 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     6241 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     6301 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     6361 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     6421 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     6481 caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     6541 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     6601 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     6661 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     6721 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     6781 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     6841 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6901 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     6961 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     7021 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     7081 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     7141 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     7201 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     7261 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     7321 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     7381 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     7441 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     7501 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     7561 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     7621 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     7681 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     7741 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     7801 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7861 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     7921 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     7981 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     8041 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     8101 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     8161 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     8221 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     8281 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     8341 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     8401 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     8461 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     8521 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     8581 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     8641 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     8701 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     8761 cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
