LOCUS       sequence-436935-        5739 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty backbone for shuttle vector construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5739)
  AUTHORS   Preston MA, Porter DF, Chen F, Buter N, Lapointe CP, Keles S, Kimble
            J, Wickens M
  TITLE     Unbiased screen of RNA tailing activities reveals a poly(UG) 
            polymerase.
  JOURNAL   Nat Methods. 2019 May;16(5):437-445. doi: 10.1038/s41592-019-0370-6.
            Epub 2019 Apr 15.
  PUBMED    30988468
REFERENCE   2  (bases 1 to 5739)
  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"; doi: 
            "10.1038/s41592-019-0370-6"; journalName: "Nat Methods"; date: 
            "2019-05"; volume: "16"; issue: "5"; pages: "437-445"
COMMENT     Sequence Label: Leu2 2u
FEATURES             Location/Qualifiers
     source          1..5739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    835..856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(871..876,877..894,895..901)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    909..925
                     /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     914..936
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     933..949
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     933..949
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             945..1259
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIYRIVLVTHH
                     "
     misc_feature    962..1018
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1019..1036)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1019..1035)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1028..1050)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1472..2818
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(2956..4050)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(4051..4456)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(4476..4494)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4562..4666
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4667..4735,4736..5527)
                     /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(4885..4904)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5698..5739,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      781 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      841 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      901 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      961 caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc tcgaattcac
     1021 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     1081 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     1141 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta
     1201 cgcatctgtg cggtatttca caccgcattt atcggatcgt acttgttacc catcattgaa
     1261 ttttgaacat ccgaacctgg gagttttccc tgaaacagat agtatatttg aacctgtata
     1321 ataatatata gtctagcgct ttacggaaga caatgtatgt atttcggttc ctggagaaac
     1381 tattgcatct attgcatagg taatcttgca cgtcgcatcc ccggttcatt ttctgcgttt
     1441 ccatcttgca cttcaatagc atatctttgt taacgaagca tctgtgcttc attttgtaga
     1501 acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac
     1561 agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt
     1621 gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt
     1681 tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct
     1741 tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt
     1801 actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag
     1861 gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc
     1921 tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat
     1981 aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa
     2041 gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt
     2101 ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg
     2161 aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat
     2221 gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag
     2281 ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt
     2341 attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg
     2401 tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagctagag
     2461 aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg
     2521 caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc
     2581 tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt
     2641 atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat attatcccat
     2701 tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct atatgctgcc
     2761 actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat attggatcga
     2821 tccgatgata agctgtcaaa catgagaatt aattctaccc tatgaacata ttccattttg
     2881 taatttcgtg tcgtttctat tatgaatttc atttataaag tttatgtaca aatatcataa
     2941 aaaaagagaa tctttttaag caaggatttt cttaacttct tcggcgacag catcaccgac
     3001 ttcggtggta ctgttggaac cacctaaatc accagttctg atacctgcat ccaaaacctt
     3061 tttaactgca tcttcaatgg ccttaccttc ttcaggcaag ttcaatgaca atttcaacat
     3121 cattgcagca gacaagatag tggcgatagg gttgacctta ttctttggca aatctggagc
     3181 agaaccgtgg catggttcgt acaaaccaaa tgcggtgttc ttgtctggca aagaggccaa
     3241 ggacgcagat ggcaacaaac ccaaggaacc tgggataacg gaggcttcat cggagatgat
     3301 atcaccaaac atgttgctgg tgattataat accatttagg tgggttgggt tcttaactag
     3361 gatcatggcg gcagaatcaa tcaattgatg ttgaaccttc aatgtaggaa attcgttctt
     3421 gatggtttcc tccacagttt ttctccataa tcttgaagag gccaaaacat tagctttatc
     3481 caaggaccaa ataggcaatg gtggctcatg ttgtagggcc atgaaagcgg ccattcttgt
     3541 gattctttgc acttctggaa cggtgtattg ttcactatcc caagcgacac catcaccatc
     3601 gtcttccttt ctcttaccaa agtaaatacc tcccactaat tctctgacaa caacgaagtc
     3661 agtaccttta gcaaattgtg gcttgattgg agataagtct aaaagagagt cggatgcaaa
     3721 gttacatggt cttaagttgg cgtacaattg aagttcttta cggattttta gtaaaccttg
     3781 ttcaggtcta acactacctg taccccattt aggaccaccc acagcaccta acaaaacggc
     3841 atcagccttc ttggaggctt ccagcgcctc atctggaagt ggaacacctg tagcatcgat
     3901 agcagcacca ccaattaaat gattttcgaa atcgaacttg acattggaac gaacatcaga
     3961 aatagcttta agaaccttaa tggcttcggc tgtgatttct tgaccaacgt ggtcacctgg
     4021 caaaacgacg atcttcttag gggcagacat tagaatggta tatccttgaa atatatatat
     4081 atattgctga aatgtaaaag gtaagaaaag ttagaaagta agacgattgc taaccaccta
     4141 ttggaaaaaa caataggtcc ttaaataata ttgtcaactt caagtattgt gatgcaagca
     4201 tttagtcatg aacgcttctc tattctatat gaaaagccgg ttccggcgct ctcacctttc
     4261 ctttttctcc caatttttca gttgaaaaag gtatatgcgt caggcgacct ctgaaattaa
     4321 caaaaaattt ccagtcatcg aatttgattc tgtgcgatag cgcccctgtg tgttctcgtt
     4381 atgttgagga aaaaaataat ggttgctaag agattcgaac tcttgcatct tacgatacct
     4441 gagtattccc acagttaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     4501 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4561 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4621 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4681 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4741 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4801 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4861 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4921 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4981 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5041 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5101 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5161 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5221 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5281 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5341 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5401 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5461 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5521 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5581 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5641 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5701 agatcctttt tttctgcgcg taatctgctg cttgcaaac
//
