LOCUS       Exported                7262 bp ds-DNA     circular SYN 05-OCT-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7262)
  AUTHORS   M. Tominaga et al
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Oct 7, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7262
                     /organism="synthetic DNA construct"
                     /lab_host="Pichia pastoris"
                     /mol_type="other DNA"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             201..1061
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                     1:  201 ..  269 / #ccffcc / signal sequence
                     2:  270 .. 1061 / #ccffcc
                     Cleavage site after base 269"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori (ColE1)
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /note="color: #31849b"
     promoter        2144..2174
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                     1: 2144 .. 2149 / #ffffff / -35
                     2: 2150 .. 2167 / #ffffff
                     3: 2168 .. 2174 / #ffffff / -10"
     protein_bind    2182..2198
                     /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)."
                     /note="color: #31849b"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     misc_feature    2241..2478
                     /label=KpARG4-UTR
                     /note="color: #a6acb3"
     CDS             2479..3876
                     /codon_start=1
                     /label=KpARG4
                     /note="color: #993366"
                     /translation="MSNQEEGLKLWGGRFTGATDPLMDLYNASLPYDKKMYKVDLEGTK
                     VYTEGLEKINLLTKDELSEIHRGLKLIEAEWAEGKFVEKPGDEDIHTANERRLGELIGR
                     GISGKVHTGRSRNDQVATDMRLYVRDNLTQLADYLKQFIQVIIKRAEQEIDVLMPGYTH
                     LQRAQPIRWSHWLSMYATYFTEDYERLNQIVKRLNKSPLGAGALAGHPYGIDREYIAER
                     LGFDSVIGNSLAAVSDRDFVVETMFWSSLFMNHISRFSEDLIIYSTGEFGFIKLADAYS
                     TGSSLMPQKKNPDSLELLRGKSGRCFGALAGFLMSIKSIPSTYNKDMQEDKEPLFDTLI
                     TVEHSILIASGVVSTLNIDAERMKNALTMDMLATDLADYLVRRGVPFRETHHISGECVR
                     QAEELNLSGIDQLSLEQLKSIDSRFEADVASTFDFEASVEKRTATGGTSKTAVLKQLDA
                     LNEKLES"
     primer_bind     3885..3918
                     /label=MT1949
                     /note="color: 黒; sequence:
                     cgacTCTAGAGGATCCatgatacgaaacgtaccg; added: 2024-08-31"
     primer_bind     3896..3958
                     /label=MT1950
                     /note="color: 黒; sequence: 
                     gatccatgatacgaaacgtaccgtatcgttaaggtAACAAGTTCAAATACCTATCTTTG
                      GCAG; added: 2024-08-31"
     promoter        3901..4192
                     /locus_tag="DAS1 promoter"
                     /label=KpPhl1.7
                     /label=DAS1 promoter
                     /note="This forward directional feature has 4 segments:
                     1: 3901 .. 3930 / #99ccff / phlO
                     2: 3931 .. 4087 / #3366ff / KpDAS1 promoter (-262_-106)
                     3: 4088 .. 4096 / #ff0000 / TATA box
                     4: 4097 .. 4192 / #3366ff / KpDAS1 promoter (-96_-1)"
     primer_bind     complement(4157..4213)
                     /label=MT1461
                     /note="color: 黒; sequence: 
                     acccttagaaaccatactagtTTTGTTCGATTATTCTCCAGATAAAATCAACAATAG; 
                     added: 2024-08-31"
     CDS             4199..4915
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                     1: 4199 .. 4201 / #05fc14
                     2: 4202 .. 4204 / #05fc14 / 1a
                     3: 4205 .. 4915 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      4993..5239
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        5286..5635
                     /label=ScTEF1
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5636..6673
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /note="color: #ccffcc"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     primer_bind     complement(6868..6884)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccgctagc catggttgtg cgacagattc actgtgaaag actgttcatt
     2281 atacccacgt ttcactggga gatgtaagcc ttaggtgttt taccctgatt agataataca
     2341 ataaccaaca gaaatacgag aatctaaact aatttcgatg attcattttt ctttttaccg
     2401 cgctgcctct tttggcaatt ctttcaccta tattctacct tctctttcct tttgttctaa
     2461 acttattacc agctatctat gtcgaatcaa gaagaaggac ttaaactgtg gggtggcagg
     2521 tttactgggg ctactgaccc cttgatggat ttgtataacg cttccttacc ttacgacaag
     2581 aaaatgtaca aggtggattt agaaggaaca aaagtttaca ctgagggcct ggagaaaatt
     2641 aatttgctaa ctaaagacga actaagtgag attcatcgtg gtctcaaatt gattgaagca
     2701 gagtgggcag aagggaagtt tgttgagaag ccaggggatg aggatattca cactgctaat
     2761 gaacgtcgct tgggtgagtt gattggtcgt ggaatctctg gtaaggttca taccggaagg
     2821 tctagaaatg atcaagttgc cactgatatg cggttgtatg tcagagacaa tctaactcag
     2881 ttggctgact atctgaagca gttcattcaa gtaatcatca agagagctga acaggaaata
     2941 gacgtcttga tgcccggtta tactcacttg caaagagctc aaccaatcag atggtctcac
     3001 tggttgagca tgtatgctac ctatttcact gaagattatg agagactgaa tcaaatcgtt
     3061 aaaaggttga acaaatcccc attgggagct ggagctttgg ctggtcatcc ttatggaatt
     3121 gatcgtgaat acattgctga gagattaggg tttgattctg ttattggtaa ttctttggcc
     3181 gctgtttcag acagagattt tgtagtcgaa accatgttct ggtcttcgtt gtttatgaat
     3241 catatttctc gattctcaga agatttgatc atttactcca ctggagagtt tggatttatc
     3301 aagttggcag atgcttattc tactggatct tctctgatgc ctcaaaaaaa aaacccagac
     3361 tctttggagt tattgagggg taaatctggt agatgttttg gggccttggc tggtttcctc
     3421 atgtctatta agtccattcc gtcaacctat aacaaagata tgcaagagga taaggagcct
     3481 ttatttgata ctctaatcac tgtagagcac tcgattttga tagcatccgg tgtagtttct
     3541 accttgaaca ttgatgccga acgaatgaag aatgctctaa ctatggatat gctggctaca
     3601 gatcttgccg actatttagt tagaagggga gttccattca gagaaactca ccacatttct
     3661 ggtgaatgtg tcagacaagc cgaggagttg aacctttctg gtattgatca gttgtccctc
     3721 gaacaattga aatccattga ctcccgtttt gaggctgatg tggcttcaac gtttgacttt
     3781 gaagccagtg ttgaaaaaag aactgccacc ggaggaactt ctaagactgc tgttttaaag
     3841 caattggatg cactgaatga aaagctagag tcttgactgc aggtcgactc tagaggatcc
     3901 atgatacgaa acgtaccgta tcgttaaggt aacaagttca aatacctatc tttggcagga
     3961 cttttcctcc tgcctttttt agcctcaggt ctcggttagc ctctaggcaa attctggtct
     4021 tcatacctat atcaactttt catcagatag cctttgggtt caaaaaagaa ctaaagcagg
     4081 atgcctgata tataaatccc agatgatctg cttttgaaac tattttcagt atcttgattc
     4141 gtttacttac aaacaactat tgttgatttt atctggagaa taatcgaaca aaactagtat
     4201 ggtttctaag ggtgaagagt tgttcactgg tgttgttcca atcttggttg agttggacgg
     4261 tgacgttaac ggacacaagt tctctgtttc tggtgaaggt gagggtgacg ctacttacgg
     4321 aaagttgact ttgaagttca tctgtactac tggtaagttg ccagttccat ggccaacttt
     4381 ggttactact ttgacttacg gtgttcagtg tttctccaga tacccagacc acatgaagca
     4441 gcacgatttc ttcaagtctg ctatgccaga gggttacgtt caagagagaa ctatcttctt
     4501 caaggacgac ggtaactaca agactagagc tgaggttaag ttcgagggtg acacattggt
     4561 taacagaatc gagttgaagg gtatcgactt caaagaggac ggaaacatct tgggtcacaa
     4621 gttggagtac aactacaact cccacaacgt ttacatcatg gctgacaagc agaagaacgg
     4681 tatcaaggtt aacttcaaga tcagacacaa catcgaggac ggttccgttc aattggctga
     4741 ccactaccaa cagaacactc caattggtga cggtccagtt ttgttgccag acaaccacta
     4801 cttgtccact caatccgctt tgtccaagga cccaaacgag aagagagatc acatggtttt
     4861 gttggagttc gttactgctg ctggtatcac tttgggtatg gacgagttgt acaagtaact
     4921 cgagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa gaccggtctt
     4981 gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt
     5041 tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct
     5101 tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg
     5161 ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag
     5221 tacagaagat taagtgagac gttcgtttgt gcaagcttag atccccgggt accgagctcg
     5281 aattcgccgt accacttcaa aacacccaag cacagcatac taaatttccc ctctttcttc
     5341 ctctagggtg tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc
     5401 gtttcttttt cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa
     5461 aatttttttt tttgattttt ttctctttcg atgacctccc attgatattt aagttaataa
     5521 acggtcttca atttctcaag tttcagtttc atttttcttg ttctattaca acttttttta
     5581 cttcttgctc attagaaaga aagcatagca atctaatcta agttttaatt acaaaatgaa
     5641 gaagccagag ttgactgcta cttccgttga gaagttcttg atcgagaagt tcgactccgt
     5701 ttccgacttg atgcaattgt ctgaaggtga agagtccaga gctttctcct ttgacgttgg
     5761 tggtagaggt tacgttttga gagttaattc ctgtgctgac ggtttctaca aggacagata
     5821 cgtttacaga cacttcgctt ccgctgcttt gccaattcca gaggttttgg acattggtga
     5881 gttctccgag tccttgactt actgtatctc cagaagagct cagggagtta cattgcaaga
     5941 cttgccagaa actgagttgc cagctgtttt gcaaccagtt gctgaagcta tggacgctat
     6001 tgctgctgct gacttgtctc aaacttctgg ttttggtcca ttcggtccac agggtattgg
     6061 tcagtacact acttggagag acttcatctg tgctatcgct gacccacatg tttaccactg
     6121 gcaaactgtt atggacgata ctgtttccgc ttccgttgct caggctttgg acgaattgat
     6181 gttgtgggct gaagattgtc ctgaggttag acatttggtt cacgctgact tcggttccaa
     6241 caacgttttg actgacaacg gtagaatcac tgctgttatc gactggtccg aagctatgtt
     6301 cggtgactct caatacgagg ttgctaacat cttcttttgg agaccatggt tggcttgtat
     6361 ggaacagcag actagatact tcgagagaag acacccagaa ttggctggtt ccccaagatt
     6421 gagagcttac atgttgagaa tcggtttgga ccagttgtac cagtccttgg ttgatggtaa
     6481 cttcgatgat gctgcttggg ctcaaggtag atgtgacgct atcgttagat ccggtgctgg
     6541 tactgttggt agaactcaga ttgctagaag atccgctgct gtttggactg acggttgtgt
     6601 tgaagttttg gctgactccg gtaacagaag accatccact agaccaagag ctaaagaggt
     6661 tggtagagtt taagtattaa gaacgttatt tatatttcaa atttttcttt tttttctcta
     6721 cagacgctgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc
     6781 tcgaagatcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6841 gggcgctctt ccgcttcctc ggaattcact ggccgtcgtt ttacaacgtc gtgactggga
     6901 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     6961 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     7021 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     7081 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     7141 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     7201 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     7261 ga
//
