LOCUS       Exported                9135 bp ds-DNA     circular SYN 26-JAN-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9135)
  AUTHORS   SnapGene License
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Jan 27, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9135
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(1..225)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(396..1256)
                     /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"
     promoter        complement(1257..1361)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1643..2807
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        3581..4285
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             4331..4351
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4367..4708
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="activation domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 activation domain
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     promoter        4714..4732
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4739..4748
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4751..4777
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    4805..5782
                     /label=Kinase 1
     misc_feature    4832..4834
                     /label=44d mutaion_GAG>AAG_E>K
     misc_feature    4859..4861
                     /label=743a_CGG>CGA_R>R
     misc_feature    4973..4975
                     /label=353g_GGG>AGG_G>R
     misc_feature    5333..5335
                     /label=12a_GAT>AAT_D>N
     terminator      5468..5470
                     /label=263g_GCG>ACG_A>T
     misc_feature    5801..5803
                     /label=896d_TGG>TAG_W>stop
     terminator      6373..6560
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     CDS             complement(6677..7771)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIEATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(7772..8177)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(8219..8235)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    8243..8259
                     /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)."
     promoter        complement(8267..8297)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8312..8333
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    8388..8421
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    8499..8532
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(8772..9135)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
       61 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
      121 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
      181 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
      241 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
      301 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
      361 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
      421 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
      481 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
      541 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
      601 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
      661 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
      721 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
      781 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
      841 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
      901 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
      961 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     1021 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1081 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1141 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1201 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     1261 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     1321 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     1381 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     1441 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     1501 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     1561 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     1621 attgtactga gagtgcacca taacgcattt aagcataaac acgcactatg ccgttcttct
     1681 catgtatata tatatacagg caacacgcag atataggtgc gacgtgaaca gtgagctgta
     1741 tgtgcgcagc tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct ttgaagttcc
     1801 tattccgaag ttcctattct ctagctagaa agtataggaa cttcagagcg cttttgaaaa
     1861 ccaaaagcgc tctgaagacg cactttcaaa aaaccaaaaa cgcaccggac tgtaacgagc
     1921 tactaaaata ttgcgaatac cgcttccaca aacattgctc aaaagtatct ctttgctata
     1981 tatctctgtg ctatatccct atataaccta cccatccacc tttcgctcct tgaacttgca
     2041 tctaaactcg acctctacat tttttatgtt tatctctagt attactcttt agacaaaaaa
     2101 attgtagtaa gaactattca tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc
     2161 tatacgtagt atatagagac aaaatagaag aaaccgttca taattttctg accaatgaag
     2221 aatcatcaac gctatcactt tctgttcaca aagtatgcgc aatccacatc ggtatagaat
     2281 ataatcgggg atgcctttat cttgaaaaaa tgcacccgca gcttcgctag taatcagtaa
     2341 acgcgggaag tggagtcagg ctttttttat ggaagagaaa atagacacca aagtagcctt
     2401 cttctaacct taacggacct acagtgcaaa aagttatcaa gagactgcat tatagagcgc
     2461 acaaaggaga aaaaaagtaa tctaagatgc tttgttagaa aaatagcgct ctcgggatgc
     2521 atttttgtag aacaaaaaag aagtatagat tctttgttgg taaaatagcg ctctcgcgtt
     2581 gcatttctgt tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta gcgctctcgc
     2641 gttgcatttt tgttttacaa aaatgaagca cagattcttc gttggtaaaa tagcgctttc
     2701 gcgttgcatt tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa aattagcgct
     2761 ctcgcgttgc atttttgttc tacaaaatga agcacagatg cttcgttgct tgcatgcctg
     2821 caggtcgaga tccgggatcg aagaaatgat ggtaaatgaa ataggaaatc aaggagcatg
     2881 aaggcaaaag acaaatataa gggtcgaacg aaaaataaag tgaaaagtgt tgatatgatg
     2941 tatttggctt tgcggcgccg aaaaaacgag tttacgcaat tgcacaatca tgctgactct
     3001 gtggcggacc cgcgctcttg ccggcccggc gataacgctg ggcgtgaggc tgtgcccggc
     3061 ggagtttttt gcgcctgcat tttccaaggt ttaccctgcg ctaaggggcg agattggaga
     3121 agcaataaga atgccggttg gggttgcgat gatgacgacc acgacaactg gtgtcattat
     3181 ttaagttgcc gaaagaacct gagtgcattt gcaacatgag tatactagaa gaatgagcca
     3241 agacttgcga gacgcgagtt tgccggtggt gcgaacaata gagcgaccat gaccttgaag
     3301 gtgagacgcg cataaccgct agagtacttt gaagaggaaa cagcaatagg gttgctacca
     3361 gtataaatag acaggtacat acaacactgg aaatggttgt ctgtttgagt acgctttcaa
     3421 ttcatttggg tgtgcacttt attatgttac aatatggaag ggaactttac acttctccta
     3481 tgcacatata ttaattaaag tccaatgcta gtagagaagg ggggtaacac ccctccgcgc
     3541 tcttttccga tttttttcta aaccgtggaa tatttcggat atccttttgt tgtttccggg
     3601 tgtacaatat ggacttcctc ttttctggca accaaaccca tacatcggga ttcctataat
     3661 accttcgttg gtctccctaa catgtaggtg gcggagggga gatatacaat agaacagata
     3721 ccagacaaga cataatgggc taaacaagac tacaccaatt acactgcctc attgatggtg
     3781 gtacataacg aactaatact gtagccctag acttgatagc catcatcata tcgaagtttc
     3841 actacccttt ttccatttgc catctattga agtaataata ggcgcatgca acttcttttc
     3901 tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat gacaaaaaaa
     3961 tgatggaaga cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt
     4021 tccagagctg atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc
     4081 acactactct ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat
     4141 aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac ctgcaattat taatcttttg
     4201 tttcctcgtc attgttctcg ttccctttct tccttgtttc tttttctgca caatatttca
     4261 agctatacca agcatacaat caactccaag ctttgcaaag atggataaag cggaattaat
     4321 tcccgagcct ccaaaaaaga agagaaaggt cgaattgggt accgccgcca attttaatca
     4381 aagtgggaat attgctgata gctcattgtc cttcactttc actaacagta gcaacggtcc
     4441 gaacctcata acaactcaaa caaattctca agcgctttca caaccaattg cctcctctaa
     4501 cgttcatgat aacttcatga ataatgaaat cacggctagt aaaattgatg atggtaataa
     4561 ttcaaaacca ctgtcacctg gttggacgga ccaaactgcg tataacgcgt ttggaatcac
     4621 tacagggatg tttaatacca ctacaatgga tgatgtatat aactatctat tcgatgatga
     4681 agatacccca ccaaacccaa aaaaagagat ctttaatacg actcactata gggcgagcgc
     4741 cgccatggag tacccatacg acgtaccaga ttacgctcat atggccatgg aggccagtga
     4801 attcatggac ggatacgagt tcgagagggc ggagatagat gcactggagc gcgtcgtacg
     4861 ggatccaaga gaaaagccaa taagcctgtc gttttggctt ctcaggcgca taacgaatga
     4921 tttctctaac gcatctgaaa ttggtcgagg tggattttcg aacgtttacc tgggggtgct
     4981 tccaagtggg ttgcgtattg ctgtcaaaaa gctttaccaa cttactgcat tggctaatga
     5041 atttgaagat gaagttctta ccacaatgag ggttgctcac aaaaacatag tgcgactcat
     5101 aggctactgt cgtcacacgc aatctgaagt cttcgaatta aaaggagaat ctattttcgc
     5161 acaggtcaga gaaaggttga tctgtacaga gtatgtgcct aatggaactc ttgatggaca
     5221 tatcactgat aagtttcatg gaggacttga ttgggaccag cgttatcaag ttctcaaagg
     5281 aatttgtcac ggtttgcatt atctgcacga tgaaatacgc gttatccaca gagatatcaa
     5341 aacagaaaat atattgctag atgataacgg cgtgccaaaa gtttttgact ttggtttttc
     5401 gaagtgcctt cctgatggag aatctttaat taaagatgaa gcaacccagg gaaccctagg
     5461 gtatttggcg ccggagtcag ttgataagca agaatattcc tttaagtccg acatatacag
     5521 ctttggtgtt gtgatcatac aattattgac tggatcgtgg atcagtaaga accgaaatgt
     5581 tgagctagaa cttaaggagt tgagaaaaag gttggtaaga gaaggagcat ttccatcatg
     5641 ggaaaacaaa taccaccaag ttagaacatg tctggagatt gtgtataact gcatggacat
     5701 cgacccagat aaaaggccga ctgcattgga aattatccaa cggcttgatg aaaccgaaag
     5761 cgcgaactat tctgttagca gtggccatgc aacaccactt tggcagtcag gagatgaaga
     5821 atccgactca gcggaagtgg atgcttttga gccagagaga acaccatcaa aagacattct
     5881 ttcagatgat gaggaacccg catcagcaga cgcaaatgga gaaacgagca cacaaaaaca
     5941 taatatgcct gatcttgtaa gtaaacagtc atgaggatcc atcgagctcg agctgcagat
     6001 gaatcgtaga tactgaaaaa ccccgcaagt tcacttcaac tgtgcatcgt gcaccatctc
     6061 aatttctttc atttatacat cgttttgcct tcttttatgt aactatactc ctctaagttt
     6121 caatcttggc catgtaacct ctgatctata gaatttttta aatgactaga attaatgccc
     6181 atcttttttt tggacctaaa ttcttcatga aaatatatta cgagggctta ttcagaagct
     6241 ttggacttct tcgccagagg tttggtcaag tctccaatca aggttgtcgg cttgtctacc
     6301 ttgccagaaa tttacgaaaa gatggaaaag ggtcaaatcg ttggtagata cgttgttgac
     6361 acttctaaat aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa
     6421 aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa attcttattc
     6481 ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagcatga ggtcgctctt
     6541 attgaccaca cctctaccgg ccggtcgaaa ttcccctacc ctatgaacat attccatttt
     6601 gtaatttcgt gtcgtttcta ttatgaattt catttataaa gtttatgtac aaatatcata
     6661 aaaaaagaga atctttttaa gcaaggattt tcttaacttc ttcggcgaca gcatcaccga
     6721 cttcggtggt actgttggaa ccacctaaat caccagttct gatacctgca tccaaaacct
     6781 ttttaactgc atcttcaatg gccttacctt cttcaggcaa gttcaatgac aatttcaaca
     6841 tcattgcagc agacaagata gtggcgatag ggttgacctt attctttggc aaatctggag
     6901 cagaaccgtg gcatggttcg tacaaaccaa atgcggtgtt cttgtctggc aaagaggcca
     6961 aggacgcaga tggcaacaaa cccaaggaac ctgggataac ggaggcttca tcggagatga
     7021 tatcaccaaa catgttgctg gtgattataa taccatttag gtgggttggg ttcttaacta
     7081 ggatcatggc ggcagaatca atcaattgat gttgaacctt caatgtagga aattcgttct
     7141 tgatggtttc ctccacagtt tttctccata atcttgaaga ggccaaaaca ttagctttat
     7201 ccaaggacca aataggcaat ggtggctcat gttgtagggc catgaaagcg gccattcttg
     7261 tgattctttg cacttctgga acggtgtatt gttcactatc ccaagcgaca ccatcaccat
     7321 cgtcttcctt tctcttacca aagtaaatac ctcccactaa ttctctgaca acaacgaagt
     7381 cagtaccttt agcaaattgt ggcttgattg gagataagtc taaaagagag tcggatgcaa
     7441 agttacatgg tcttaagttg gcgtacaatt gaagttcttt acggattttt agtaaacctt
     7501 gttcaggtct aacactacct gtaccccatt taggaccacc cacagcacct aacaaaacgg
     7561 catcagcctt cttggaggct tccagcgcct catctggaag tgggacacct gtagcttcga
     7621 tagcagcacc accaattaaa tgattttcga aatcgaactt gacattggaa cgaacatcag
     7681 aaatagcttt aagaacctta atggcttcgg ctgtgatttc ttgaccaacg tggtcacctg
     7741 gcaaaacgac gatcttctta ggggcagaca ttagaatggt atatccttga aatatatata
     7801 tatattgctg aaatgtaaaa ggtaagaaaa gttagaaagt aagacgattg ctaaccacct
     7861 attggaaaaa acaataggtc cttaaataat attgtcaact tcaagtattg tgatgcaagc
     7921 atttagtcat gaacgcttct ctattctata tgaaaagccg gttccggcgc tctcaccttt
     7981 cctttttctc ccaatttttc agttgaaaaa ggtatatgcg tcaggcgacc tctgaaatta
     8041 acaaaaaatt tccagtcatc gaatttgatt ctgtgcgata gcgcccctgt gtgttctcgt
     8101 tatgttgagg aaaaaaataa tggttgctaa gagattcgaa ctcttgcatc ttacgatacc
     8161 tgagtattcc cacagttggg ggatctcgac tctagctaga ggatcaattc gtaatcatgt
     8221 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     8281 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     8341 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgata acttcgtata
     8401 atgtatgcta tacgaagtta ttaggtctga agaggagttt acgtccagcc aagctagctt
     8461 ggctgcaggt cgagcggccg cgatccggaa cccttaatat aacttcgtat aatgtatgct
     8521 atacgaagtt atcagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     8581 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     8641 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     8701 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     8761 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     8821 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     8881 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     8941 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     9001 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     9061 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     9121 gcagccactg gtaac
//
