LOCUS       40924_38013        7741 bp DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7741)
  AUTHORS   Stefan CJ, Audhya A, Emr SD
  TITLE     The yeast synaptojanin-like proteins control the cellular 
            distribution of phosphatidylinositol (4,5)-bisphosphate.
  JOURNAL   Mol Biol Cell. 2002 Feb;13(2):542-57.
  PUBMED    11854411
REFERENCE   2  (bases 1 to 7741)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7741)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Biol
            Cell."; date: "2002-02"; volume: "13(2)"; pages: "542-57"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7741
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    91..112
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        127..157
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    165..181
                     /label=lac operator
                     /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     189..205
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        226..244
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     281..297
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             793..1506
                     /codon_start=1
                     /label=GFP (S65T)
                     /note="S65T variant of Aequorea victoria green fluorescent 
                     protein (Heim et al., 1995)"
                     /translation="MGKGVELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     promoter        complement(2399..2417)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2427..2443)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      2584..3039
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(3173..3973)
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(3974..4189)
                     /label=URA3 promoter
     primer_bind     complement(4213..4232)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     4332..4354
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4392..4410)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     rep_origin      4451..5793
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     promoter        5820..5924
                     /label=AmpR promoter
     CDS             5925..6782
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6956..7544
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7698..7715
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
       61 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
      121 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
      181 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
      241 aagggaacaa aagctggagc tccaccgcgg tggcggccgc tctagaacta gtggatcctt
      301 ctgcacaaga agccatattg acagagcagt atgtgaggac ctttcttcta cacaatgtaa
      361 cagtcaatgg ttgtagtcaa taccacgacc tttacgtgca gttttagagc gaaacttcgg
      421 ttttttgaga cttacctcgt gtttgtgtct ccctgggaat ggagccgcgc cacccttgtc
      481 tattatgtat agtcttattt aatcatagcg atgttggtca tccagtacac ttcggtagca
      541 accttcgttt gtgattgtct tggtaattgc ttccaacaac tttatccatc attgagacag
      601 gggccatatc acccgcgggg tctcaaagaa ggggcccact aataaaagca cgagataaga
      661 atgccagcaa aaaagctccg aaataattct tttcgtcttc cctcctagtc ttaacaagac
      721 aagagagaga gagagaaaga atactcacta gagattgttt cttttctact caacttaaag
      781 tatacatacg ctatgggtaa aggtgtagaa cttttcactg gagttgtccc aattcttgtt
      841 gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat
      901 gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca
      961 tggccaacac ttgtcactac ttttacgtat ggtgttcaat gcttttcaag atacccagat
     1021 catatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga
     1081 actatatttt tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt
     1141 gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt
     1201 cttggacaca aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa
     1261 caaaagaatg gaatcaaagt taacttcaaa attagacaca acattgaaga tggaagcgtt
     1321 caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca
     1381 gacaaccatt acctgtccac acaatctgcc ctttcgaaag atcccaacga aaagagagac
     1441 cacatggtcc ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta
     1501 tacaagtccg gactcagatc ccacgggctc caggatgacc cggaccttca ggcccttctg
     1561 aagggcagcc agcttctgaa ggtgaagtcc agctcgtggc gtagggaacg cttctacaag
     1621 ctacaggagg actgcaagac catctggcag gaatctcgaa aggtcatgag gtccccggag
     1681 tcgcagctgt tctccatcga ggacattcag gaggtacgga tgggacaccg cacagaaggc
     1741 ctggagaagt ttgcccgaga catccccgag gatcgatgct tctccattgt cttcaaggac
     1801 cagcgcaaca ccctagacct cattgcccca tcaccagctg acgctcagca ctgggtgcag
     1861 ggcctgcgca agatcatcca ccactccggc tccatggacc agcggcagaa ggcagcggcc
     1921 gcaggattcc gcaagggcga attcgccctt gcggaatccg ccgcggcagc tcacgggctc
     1981 caggatgacc cggaccttca ggcccttctg aagggcagcc agcttctgaa ggtgaagtcc
     2041 agctcgtggc gtagggaacg cttctacaag ctacaggagg actgcaagac catctggcag
     2101 gaatctcgaa aggtcatgag gtccccggag tcgcagctgt tctccatcga ggacattcag
     2161 gaggtacgga tgggacaccg cacagaaggc ctggagaagt ttgcccgaga catccccgag
     2221 gatcgatgct tctccattgt cttcaaggac cagcgcaaca ccctagacct cattgcccca
     2281 tcaccagctg acgctcagca ctgggtgcag ggcctgcgca agatcatcca ccactccggc
     2341 tccatggacc agcggcagaa gtagtcgacc tcgagggggg gcccggtacc caattcgccc
     2401 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     2461 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     2521 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     2581 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2641 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2701 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2761 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2821 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2881 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2941 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3001 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct
     3061 tacgcatctg tgcggtattt cacaccgcat agggtaataa ctgatataat taaattgaag
     3121 ctctaatttg tgagtttagt atacatgcat ttacttataa tacagttttt tagttttgct
     3181 ggccgcatct tctcaaatat gcttcccagc ctgcttttct gtaacgttca ccctctacct
     3241 tagcatccct tccctttgca aatagtcctc ttccaacaat aataatgtca gatcctgtag
     3301 agaccacatc atccacggtt ctatactgtt gacccaatgc gtctcccttg tcatctaaac
     3361 ccacaccggg tgtcataatc aaccaatcgt aaccttcatc tcttccaccc atgtctcttt
     3421 gagcaataaa gccgataaca aaatctttgt cgctcttcgc aatgtcaaca gtacccttag
     3481 tatattctcc agtagatagg gagcccttgc atgacaattc tgctaacatc aaaaggcctc
     3541 taggttcctt tgttacttct tctgccgcct gcttcaaacc gctaacaata cctgggccca
     3601 ccacaccgtg tgcattcgta atgtctgccc attctgctat tctgtataca cccgcagagt
     3661 actgcaattt gactgtatta ccaatgtcag caaattttct gtcttcgaag agtaaaaaat
     3721 tgtacttggc ggataatgcc tttagcggct taactgtgcc ctccatggaa aaatcagtca
     3781 agatatccac atgtgttttt agtaaacaaa ttttgggacc taatgcttca actaactcca
     3841 gtaattcctt ggtggtacga acatccaatg aagcacacaa gtttgtttgc ttttcgtgca
     3901 tgatattaaa tagcttggca gcaacaggac taggatgagt agcagcacgt tccttatatg
     3961 tagctttcga catgatttat cttcgtttcc tgcaggtttt tgttctgtgc agttgggtta
     4021 agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat accaatctaa
     4081 gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca aaaaaatttc
     4141 aaggaaaccg aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaaa aggtggtatg
     4201 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     4261 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4321 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4381 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     4441 ttcttagtat gatccaatat caaaggaaat gatagcattg aaggatgaga ctaatccaat
     4501 tgaggagtgg cagcatatag aacagctaaa gggtagtgct gaaggaagca tacgataccc
     4561 cgcatggaat gggataatat cacaggaggt actagactac ctttcatcct acataaatag
     4621 acgcatataa gtacgcattt aagcataaac acgcactatg ccgttcttct catgtatata
     4681 tatatacagg caacacgcag atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc
     4741 tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct ttgaagttcc tattccgaag
     4801 ttcctattct ctagaaagta taggaacttc agagcgcttt tgaaaaccaa aagcgctctg
     4861 aagacgcact ttcaaaaaac caaaaacgca ccggactgta acgagctact aaaatattgc
     4921 gaataccgct tccacaaaca ttgctcaaaa gtatctcttt gctatatatc tctgtgctat
     4981 atccctatat aacctaccca tccacctttc gctccttgaa cttgcatcta aactcgacct
     5041 ctacattttt tatgtttatc tctagtatta ctctttagac aaaaaaattg tagtaagaac
     5101 tattcataga gtgaatcgaa aacaatacga aaatgtaaac atttcctata cgtagtatat
     5161 agagacaaaa tagaagaaac cgttcataat tttctgacca atgaagaatc atcaacgcta
     5221 tcactttctg ttcacaaagt atgcgcaatc cacatcggta tagaatataa tcggggatgc
     5281 ctttatcttg aaaaaatgca cccgcagctt cgctagtaat cagtaaacgc gggaagtgga
     5341 gtcaggcttt ttttatggaa gagaaaatag acaccaaagt agccttcttc taaccttaac
     5401 ggacctacag tgcaaaaagt tatcaagaga ctgcattata gagcgcacaa aggagaaaaa
     5461 aagtaatcta agatgctttg ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca
     5521 aaaaagaagt atagattctt tgttggtaaa atagcgctct cgcgttgcat ttctgttctg
     5581 taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt
     5641 ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg
     5701 ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt
     5761 ttgttctaca aaatgaagca cagatgcttc gttcaggtgg cacttttcgg ggaaatgtgc
     5821 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5881 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5941 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6001 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6061 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6121 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6181 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6241 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6301 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6361 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6421 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6481 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6541 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6601 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6661 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6721 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6781 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6841 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6901 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6961 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7021 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7081 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     7141 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7201 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7261 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7321 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7381 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7441 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7501 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7561 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     7621 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     7681 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     7741 a
//
