LOCUS       Exported                7033 bp ds-DNA     circular SYN 26-JUL-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7033)
  AUTHORS   Lewis Lab
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Dec 16, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7033
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        127..231
                     /gene="bla"
                     /label=AmpR promoter
     CDS             232..1092
                     /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"
     rep_origin      1263..1851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    1907..2267
                     /label=pGAL7 promoter
     misc_feature    2311..2408
                     /label=Retron EC86
     misc_feature    2424..2524
                     /label=ADE2 repair template
                     /note="Introduces frameshift mutation"
     misc_feature    2474
                     /label=Frameshift insertion
                     /note="""C"" inserted to introduce frameshift, located in 
                     the PAM seq."
     misc_feature    2525..2558
                     /label=MSD 3'
     misc_feature    2559..2578
                     /label=ADE2 gRNA
                     /note="Targets cas9 to cut at nt 450 of ADE2 gene"
     misc_feature    2579..2740
                     /label=sgRNA and HDV
                     /note="structural guide RNA and HDV ribozyme"
     misc_feature    2741..3099
                     /label=tGAL7
     promoter        complement(3113..3131)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3141..3157)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3272..3615
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             3616..4425
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      4431..4628
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      4745..5200
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(5331..6134)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6135..6350)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     misc_feature    join(6620..7033,1..90)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 ttttctttaa tttctttttt tactttctat ttttaattta tatatttata ttaaaaaatt
       61 taaattataa ttatttttat agcacgtgat gaaaaggacc caggtggcac ttttcgggga
      121 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
      181 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
      241 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
      301 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
      361 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
      421 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
      481 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
      541 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
      601 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
      661 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
      721 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
      781 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
      841 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
      901 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
      961 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     1021 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     1081 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     1141 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     1201 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1261 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     1321 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     1381 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     1441 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     1501 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     1561 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     1621 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     1681 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     1741 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     1801 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     1861 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcaagt agagggggta
     1921 atttttcccc tttattttgt tcatacattc ttaaattgct ttgcctctcc ttttggaaag
     1981 ctatacttcg gagcactgtt gagcgaaggc tcattagata tattttctgt cattttcctt
     2041 aacccaaaaa taagggaaag ggtccaaaaa gcgctcggac aactgttgac cgtgatccga
     2101 aggactggct atacagtgtt cacaaaatag ccaagctgaa aataatgtgt agctatgttc
     2161 agttagtttg gctagcaaag atataaaagc aggtcggaaa tatttatggg cattattatg
     2221 cagagcatca acatgataaa aaaaaacagt tgaatattcc ctcaaaaggg tgcgcatctg
     2281 atgagtccgt gaggacgaaa cgagctagct cgtcatgcgc acccttagcg agaggtttat
     2341 cattaaggtc aacctctgga tgttgtttcg gcatcctgca ttgaatctga gttactgtct
     2401 gttttcctgg gtcacgcgta ggagggtttt ccattcgtct tgaagtcgag gactttggca
     2461 tacgatggaa gagcgtaact tcgttgtaaa gaataaggaa atgattccgg aagctttgga
     2521 agtaaggaaa cccgtttctt ctgacgtaag ggtgcgcaac tttggcatac gatggaaggt
     2581 ttcagagcta tgctggaaac agcatagcaa gttgaaataa ggctagtccg ttatcaactt
     2641 gaaaaagtgg caccgagtcg gtgctttttg atggccggca tggtcccagc ctcctcgctg
     2701 gcgccggctg ggcaacacct tcgggtggcg aatgggactt aaagaaagtg gaatattcat
     2761 tcatatcata ttttttctat taactgcctg gtttctttta aattttttat tggttgtcga
     2821 cttgaacgga gtgacaatat atatatatat atatttaata atgacatcat tatctgtaaa
     2881 tctgattctt aatgctattc tagttatgta agagtggtcc tttccataaa aaaaaaaaaa
     2941 aagaaaaaag aattttagga atacaatgca gcttgtaagt aaaatctgga atattcatat
     3001 cgccacaact tcttatgctt ataaaagcac taatgcctga atttatgttg aaaatatgtg
     3061 tcacaaataa agaaactgtg acatctgaca catttccacg tacccaattc gccctatagt
     3121 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     3181 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccaggat ctgtttagct
     3241 tgcctcgtcc ccgccgggtc acccggccag cgacatggag gcccagaata ccctccttga
     3301 cagtcttgac gtgcgcagct caggggcatg atgtgactgt cgcccgtaca tttagcccat
     3361 acatccccat gtataatcat ttgcatccat acattttgat ggccgcacgg cgcgaagcaa
     3421 aaattacggc tcctcgctgc agacctgcga gcagggaaac gctcccctca cagacgcgtt
     3481 gaattgtccc cacgccgcgc ccctgtagag aaatataaaa ggttaggatt tgccactgag
     3541 gttcttcttt catatacttc cttttaaaat cttgctagga tacagttctc acatcacatc
     3601 cgaacataaa caaccatggg taaggaaaag actcacgttt cgaggccgcg attaaattcc
     3661 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     3721 gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     3781 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     3841 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     3901 accactgcga tccccggcaa aacagcattc caggtattag aagaatatcc tgattcaggt
     3961 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     4021 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     4081 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     4141 gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt cactcatggt
     4201 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     4261 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     4321 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     4381 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcagt actgacaata
     4441 aaaagattct tgttttcaag aacttgtcat ttgtatagtt tttttatatt gtagttgttc
     4501 tattttaatc aaatgttagc gtgatttata ttttttttcg cctcgacatc atctgcccag
     4561 atgcgaagtt aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg
     4621 ctatactgct gtcgattcga tactaacgcc gccatccagt gtcgaaaacg ctggcgtaat
     4681 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     4741 cgcgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4801 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4861 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4921 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     4981 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5041 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     5101 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     5161 tttaacgcga attttaacaa aatattaacg tttacaattt cctgatgcgg tattttctcc
     5221 ttacgcatct gtgcggtatt tcacaccgca tagggtaata actgatataa ttaaattgaa
     5281 gctctaattt gtgagtttag tatacatgca tttacttata atacagtttt ttagttttgc
     5341 tggccgcatc ttctcaaata tgcttcccag cctgcttttc tgtaacgttc accctctacc
     5401 ttagcatccc ttccctttgc aaatagtcct cttccaacaa taataatgtc agatcctgta
     5461 gagaccacat catccacggt tctatactgt tgacccaatg cgtctccctt gtcatctaaa
     5521 cccacaccgg gtgtcataat caaccaatcg taaccttcat ctcttccacc catgtctctt
     5581 tgagcaataa agccgataac aaaatctttg tcgctcttcg caatgtcaac agtaccctta
     5641 gtatattctc cagtagatag ggagcccttg catgacaatt ctgctaacat caaaaggcct
     5701 ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac cgctaacaat acctgggccc
     5761 accacaccgt gtgcattcgt aatgtctgcc cattctgcta ttctgtatac acccgcagag
     5821 tactgcaatt tgactgtatt accaatgtca gcaaattttc tgtcttcgaa gagtaaaaaa
     5881 ttgtacttgg cggataatgc ctttagcggc ttaactgtgc cctccatgga aaaatcagtc
     5941 aagatatcca catgtgtttt tagtaaacaa attttgggac ctaatgcttc aactaactcc
     6001 agtaattcct tggtggtacg aacatccaat gaagcacaca agtttgtttg cttttcgtgc
     6061 atgatattaa atagcttggc agcaacagga ctaggatgag tagcagcacg ttccttatat
     6121 gtagctttcg acatgattta tcttcgtttc ctgcaggttt ttgttctgtg cagttgggtt
     6181 aagaatactg ggcaatttca tgtttcttca acactacata tgcgtatata taccaatcta
     6241 agtctgtgct ccttccttcg ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt
     6301 caaagaaacc gaaatcaaaa aaaagaataa aaaaaaaatg atgaattgaa ttgaaaagct
     6361 gtggtatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     6421 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6481 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6541 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     6601 ataatggttt cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa
     6661 tgatggaata atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat
     6721 tttagaaagt aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa
     6781 ggtttaaaaa atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc
     6841 agattaaata gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg
     6901 tgtgtggtct tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa
     6961 gagcaagata aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa
     7021 aacaaaaact att
//
