LOCUS       Exported                5110 bp ds-DNA     circular SYN 26-FEB-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5110)
  AUTHORS   Arthur
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Feb 26, 2019 from SnapGene 4.3.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5110
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        2..937
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     regulatory      949..958
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             955..1947
                     /codon_start=1
                     /label=mouseGIRK2
                     /translation="MAKRKIQRYVRKDGKCNVHHGNVRETYRYLTDIFTTLVDLKWRFN
                     LLIFVMVYTVTWLFFGMIWWLIAYIRGDMDHIEDPSWTPCVTNLNGFVSAFLFSIETET
                     TIGYGYRVITDKCPEGIILLLIQSVLGSIVNAFMVGCMFVKISQPKKRAETLVFSTHAV
                     ISMRDGKLCLMFRVGDLRNSHIVEASIRAKLIKSKQTSEGEFIPLNQSDINVGYYTGDD
                     RLFLVSPLIISHEINQQSPFWEISKAQLPKEELEIVVILEGIVEATGMTCQARSSYITS
                     EILWGYRFTPVLTMEDGFYEVDYNSFHETYETSTPSLSAKELAELANRAE"
     CDS             1957..1977
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2002..2715
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             2734..2757
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2764..2781
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2792..2821
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2837..2854
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2934..3180
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3195..3606
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3614..3661
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3680..4054
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      4120..4367
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(4442..5030)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag
       61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt
      121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc
      181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta
      241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta
      301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
      361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
      421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
      481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt
      541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
      601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
      661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
      721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
      781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
      841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
      901 caacttgaga agatcaaaaa acaactaatt attcgaagct agcctcgagc caccatggca
      961 aaacgtaaga tccaacgtta cgttcgtaag gacggtaagt gcaatgtgca tcatggtaat
     1021 gtaagagaaa cctacagata cctaacggac attttcacca ctttggtgga tttgaaatgg
     1081 cgtttcaact tattgatatt tgttatggta tacaccgtta cgtggctatt tttcggtatg
     1141 atttggtggc taattgcata tataagaggt gacatggatc acatcgaaga cccctcctgg
     1201 actccatgtg ttaccaatct aaacggcttt gtttccgcat tcttgttttc catcgaaact
     1261 gagaccacaa ttggttacgg ttaccgtgta attacagata aatgcccaga gggtatcatc
     1321 ttgttactaa ttcaatccgt tttaggctcc attgttaatg ctttcatggt cggttgcatg
     1381 tttgtgaaaa tttctcagcc caaaaagagg gctgaaactc ttgttttctc cacacatgca
     1441 gttatatcaa tgagggatgg caagttatgc ctgatgtttc gtgtcggaga cctaagaaat
     1501 tcacatatag tggaagcaag tattagagct aaactgataa aaagtaagca aacatccgag
     1561 ggcgagttca tcccattgaa tcagtctgac attaacgtgg gatactatac tggcgacgac
     1621 cgtctttttt tagttagtcc acttatcatt tcccacgaaa ttaatcagca gtcccctttt
     1681 tgggagattt ccaaggctca attgcctaag gaagaactag agattgtcgt gattcttgaa
     1741 ggcatcgtcg aagccactgg catgacgtgc caagcacgtt cctcttacat cacatccgag
     1801 atactatggg gatatagatt cacgcccgtc ttaaccatgg aagatggatt ctacgaagta
     1861 gattataaca gtttccatga gacctatgag acgagtactc cttccctatc tgcaaaggaa
     1921 ttggctgaac tggctaatag agccgagtcg aattctgaaa acctgtattt tcagggttcc
     1981 ggtagcactg ctgccgccgc tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     2041 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     2101 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     2161 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     2221 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2281 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2341 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2401 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     2461 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     2521 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2581 ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag
     2641 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     2701 gacgagctgt acaagtccgg aggtggtgca agctggagcc acccgcagtt cgaaaagggt
     2761 agccaccacc accatcatca ctaatagtct agaacaaaaa ctcatctcag aagaggatct
     2821 gaatagcgcc gtcgaccatc atcatcatca tcattgagtt tgtagcctta gacatgactg
     2881 ttcctcagtt caagttgggc acttacgaga agaccggtct tgctagattc taatcaagag
     2941 gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg
     3001 taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc
     3061 tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg
     3121 agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga
     3181 ccttcgtttg tgcggatccc ccacacacca tagcttcaaa atgtttctac tcctttttta
     3241 ctcttccaga ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca cccaagcaca
     3301 gcatactaaa ttttccctct ttcttcctct agggtgtcgt taattacccg tactaaaggt
     3361 ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag gcaataaaaa
     3421 tttttatcac gtttcttttt cttgaaattt ttttttttag tttttttctc tttcagtgac
     3481 ctccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt
     3541 cttgttctat tacaactttt tttacttctt gttcattaga aagaaagcat agcaatctaa
     3601 tctaaggggc ggtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     3661 caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     3721 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     3781 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     3841 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     3901 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     3961 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     4021 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtc cgacggcggc ccacgggtcc
     4081 caggcctcgg agatccgtcc cccttttcct ttgtcgatat catgtaatta gttatgtcac
     4141 gcttacattc acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac
     4201 ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta
     4261 tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga
     4321 aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag ctggagacca
     4381 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4441 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4501 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4561 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4621 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4681 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4741 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4801 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4861 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4921 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4981 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5041 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5101 tcatgagatc
//
