LOCUS       Exported                7269 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 7269)
  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..7269
                     /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    2436..2536
                     /label=ADE2 repair template
                     /note="Introduces frameshift"
     misc_feature    2486
                     /label=Frameshift insertion
                     /note="""C"" inserted to introduce frameshift, located in 
                     the PAM seq."
     misc_feature    2537..2570
                     /label=MSD 3'
     misc_feature    2571..2590
                     /label=ADE2 gRNA
                     /note="Targets cas9 to cleave at nt 450 in ADE2 gene"
     misc_feature    2591..2752
                     /label=sgRNA and HDV
                     /note="structural guide RNA and HDV ribozyme"
     misc_feature    2753..3111
                     /label=tGAL7
     promoter        complement(3125..3143)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3153..3169)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      3291..3488
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3494..4522)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     promoter        complement(4523..4866)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      4981..5436
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     CDS             complement(5567..6370)
                     /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(6371..6586)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     misc_feature    join(6856..7269,1..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 gttttccttc cttctgtacc gggtcacgcg taggagggtt ttccattcgt cttgaagtcg
     2461 aggactttgg catacgatgg aagagcgtaa cttcgttgta aagaataagg aaatgattcc
     2521 ggaagctttg gaagtaagga aacccgtttc ttctgacgta agggtgcgca actttggcat
     2581 acgatggaag gtttcagagc tatgctggaa acagcatagc aagttgaaat aaggctagtc
     2641 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tgatggccgg catggtccca
     2701 gcctcctcgc tggcgccggc tgggcaacac cttcgggtgg cgaatgggac ttaaagaaag
     2761 tggaatattc attcatatca tattttttct attaactgcc tggtttcttt taaatttttt
     2821 attggttgtc gacttgaacg gagtgacaat atatatatat atatatttaa taatgacatc
     2881 attatctgta aatctgattc ttaatgctat tctagttatg taagagtggt cctttccata
     2941 aaaaaaaaaa aaaagaaaaa agaattttag gaatacaatg cagcttgtaa gtaaaatctg
     3001 gaatattcat atcgccacaa cttcttatgc ttataaaagc actaatgcct gaatttatgt
     3061 tgaaaatatg tgtcacaaat aaagaaactg tgacatctga cacatttcca cgtacccaat
     3121 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     3181 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagt
     3241 cgagagctcg ttttcgacac tggatggcgg cgttagtatc gaatcgacag cagtatagcg
     3301 accagcattc acatacgatt gacgcatgat attactttct gcgcacttaa cttcgcatct
     3361 gggcagatga tgtcgaggcg aaaaaaaata taaatcacgc taacatttga ttaaaataga
     3421 acaactacaa tataaaaaaa ctatacaaat gacaagttct tgaaaacaag aatcttttta
     3481 ttgtcagtac tgattattcc tttgccctcg gacgagtgct ggggcgtcgg tttccactat
     3541 cggcgagtac ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg
     3601 tacgcccgac agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag
     3661 ctgcatcatc gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga
     3721 gcatatacgc ccggagccgc ggcgatcctg caagctccgg atgcctccgc tcgaagtagc
     3781 gcgtctgctg ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt
     3841 attgggaatc cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt
     3901 ccgtcaggac attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt
     3961 cctcggccca aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt
     4021 ccatcacagt ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc
     4081 atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa
     4141 gatcggccgc agcaatggca tccattgcct ccgcgaccgg ctgcagaaca gcgggcagtt
     4201 cggtttcagg caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca
     4261 ggctctcgct gaattcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa
     4321 agtgccgata aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga
     4381 catatccacg ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct
     4441 gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg
     4501 tgagttcagg ctttttaccc atggttgttt atgttcggat gtgatgtgag aactgtatcc
     4561 tagcaagatt ttaaaaggaa gtatatgaaa gaagaacctc agtggcaaat cctaaccttt
     4621 tatatttctc tacaggggcg cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt
     4681 tccctgctcg caggtctgca gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc
     4741 aaaatgtatg gatgcaaatg attatacatg gggatgtatg ggctaaatgt acgggcgaca
     4801 gtcacatcat gcccctgagc tgcgcacgtc aagactgtca aggagggtat tctgggcctc
     4861 catgtcgctg gccgggtgac ccggcgggga cgaggcaagc taaacactgg cgtaatagcg
     4921 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcg
     4981 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5041 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5101 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5161 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5221 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5281 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5341 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5401 acgcgaattt taacaaaata ttaacgttta caatttcctg atgcggtatt ttctccttac
     5461 gcatctgtgc ggtatttcac accgcatagg gtaataactg atataattaa attgaagctc
     5521 taatttgtga gtttagtata catgcattta cttataatac agttttttag ttttgctggc
     5581 cgcatcttct caaatatgct tcccagcctg cttttctgta acgttcaccc tctaccttag
     5641 catcccttcc ctttgcaaat agtcctcttc caacaataat aatgtcagat cctgtagaga
     5701 ccacatcatc cacggttcta tactgttgac ccaatgcgtc tcccttgtca tctaaaccca
     5761 caccgggtgt cataatcaac caatcgtaac cttcatctct tccacccatg tctctttgag
     5821 caataaagcc gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta cccttagtat
     5881 attctccagt agatagggag cccttgcatg acaattctgc taacatcaaa aggcctctag
     5941 gttcctttgt tacttcttct gccgcctgct tcaaaccgct aacaatacct gggcccacca
     6001 caccgtgtgc attcgtaatg tctgcccatt ctgctattct gtatacaccc gcagagtact
     6061 gcaatttgac tgtattacca atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt
     6121 acttggcgga taatgccttt agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga
     6181 tatccacatg tgtttttagt aaacaaattt tgggacctaa tgcttcaact aactccagta
     6241 attccttggt ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga
     6301 tattaaatag cttggcagca acaggactag gatgagtagc agcacgttcc ttatatgtag
     6361 ctttcgacat gatttatctt cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga
     6421 atactgggca atttcatgtt tcttcaacac tacatatgcg tatatatacc aatctaagtc
     6481 tgtgctcctt ccttcgttct tccttctgtt cggagattac cgaatcaaaa aaatttcaaa
     6541 gaaaccgaaa tcaaaaaaaa gaataaaaaa aaaatgatga attgaattga aaagctgtgg
     6601 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     6661 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     6721 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     6781 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     6841 tggtttctta ggacggatcg cttgcctgta acttacacgc gcctcgtatc ttttaatgat
     6901 ggaataattt gggaatttac tctgtgttta tttattttta tgttttgtat ttggatttta
     6961 gaaagtaaat aaagaaggta gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt
     7021 taaaaaattt caacaaaaag cgtactttac atatatattt attagacaag aaaagcagat
     7081 taaatagata tacattcgat taacgataag taaaatgtaa aatcacagga ttttcgtgtg
     7141 tggtcttcta cacagacaag atgaaacaat tcggcattaa tacctgagag caggaagagc
     7201 aagataaaag gtagtatttg ttggcgatcc ccctagagtc ttttacatct tcggaaaaca
     7261 aaaactatt
//
