LOCUS       Exported                7232 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 7232)
  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..7232
                     /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..3954
                     /label=MT1951
                     /note="color: 黒; sequence: 
                     gatccatgatacgaaacgtaccgtatcgttaaggtCTTTTCCTCCTGCCTTTTTTAGCC
                      ; added: 2024-08-31"
     promoter        3901..4162
                     /locus_tag="DAS1 promoter"
                     /label=KpPhl1.8
                     /label=DAS1 promoter
                     /note="This forward directional feature has 4 segments:
                     1: 3901 .. 3930 / #99ccff / phlO
                     2: 3931 .. 4057 / #3366ff / KpDAS1 promoter (-232_-106)
                     3: 4058 .. 4066 / #ff0000 / TATA box
                     4: 4067 .. 4162 / #3366ff / KpDAS1 promoter (-96_-1)"
     primer_bind     complement(4127..4183)
                     /label=MT1461
                     /note="color: 黒; sequence: 
                     acccttagaaaccatactagtTTTGTTCGATTATTCTCCAGATAAAATCAACAATAG; 
                     added: 2024-08-31"
     CDS             4169..4885
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                     1: 4169 .. 4171 / #05fc14
                     2: 4172 .. 4174 / #05fc14 / 1a
                     3: 4175 .. 4885 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      4963..5209
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        5256..5605
                     /label=ScTEF1
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5606..6643
                     /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(6838..6854)
                     /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 cttttcctcc tgcctttttt agcctcaggt
     3961 ctcggttagc ctctaggcaa attctggtct tcatacctat atcaactttt catcagatag
     4021 cctttgggtt caaaaaagaa ctaaagcagg atgcctgata tataaatccc agatgatctg
     4081 cttttgaaac tattttcagt atcttgattc gtttacttac aaacaactat tgttgatttt
     4141 atctggagaa taatcgaaca aaactagtat ggtttctaag ggtgaagagt tgttcactgg
     4201 tgttgttcca atcttggttg agttggacgg tgacgttaac ggacacaagt tctctgtttc
     4261 tggtgaaggt gagggtgacg ctacttacgg aaagttgact ttgaagttca tctgtactac
     4321 tggtaagttg ccagttccat ggccaacttt ggttactact ttgacttacg gtgttcagtg
     4381 tttctccaga tacccagacc acatgaagca gcacgatttc ttcaagtctg ctatgccaga
     4441 gggttacgtt caagagagaa ctatcttctt caaggacgac ggtaactaca agactagagc
     4501 tgaggttaag ttcgagggtg acacattggt taacagaatc gagttgaagg gtatcgactt
     4561 caaagaggac ggaaacatct tgggtcacaa gttggagtac aactacaact cccacaacgt
     4621 ttacatcatg gctgacaagc agaagaacgg tatcaaggtt aacttcaaga tcagacacaa
     4681 catcgaggac ggttccgttc aattggctga ccactaccaa cagaacactc caattggtga
     4741 cggtccagtt ttgttgccag acaaccacta cttgtccact caatccgctt tgtccaagga
     4801 cccaaacgag aagagagatc acatggtttt gttggagttc gttactgctg ctggtatcac
     4861 tttgggtatg gacgagttgt acaagtaact cgagccttag acatgactgt tcctcagttc
     4921 aagttgggca cttacgagaa gaccggtctt gctagattct aatcaagagg atgtcagaat
     4981 gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt aacctatata
     5041 gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct gatcagccta
     5101 tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt
     5161 tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagac gttcgtttgt
     5221 gcaagcttag atccccgggt accgagctcg aattcgccgt accacttcaa aacacccaag
     5281 cacagcatac taaatttccc ctctttcttc ctctagggtg tcgttaatta cccgtactaa
     5341 aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata
     5401 aaaattttta tcacgtttct ttttcttgaa aatttttttt tttgattttt ttctctttcg
     5461 atgacctccc attgatattt aagttaataa acggtcttca atttctcaag tttcagtttc
     5521 atttttcttg ttctattaca acttttttta cttcttgctc attagaaaga aagcatagca
     5581 atctaatcta agttttaatt acaaaatgaa gaagccagag ttgactgcta cttccgttga
     5641 gaagttcttg atcgagaagt tcgactccgt ttccgacttg atgcaattgt ctgaaggtga
     5701 agagtccaga gctttctcct ttgacgttgg tggtagaggt tacgttttga gagttaattc
     5761 ctgtgctgac ggtttctaca aggacagata cgtttacaga cacttcgctt ccgctgcttt
     5821 gccaattcca gaggttttgg acattggtga gttctccgag tccttgactt actgtatctc
     5881 cagaagagct cagggagtta cattgcaaga cttgccagaa actgagttgc cagctgtttt
     5941 gcaaccagtt gctgaagcta tggacgctat tgctgctgct gacttgtctc aaacttctgg
     6001 ttttggtcca ttcggtccac agggtattgg tcagtacact acttggagag acttcatctg
     6061 tgctatcgct gacccacatg tttaccactg gcaaactgtt atggacgata ctgtttccgc
     6121 ttccgttgct caggctttgg acgaattgat gttgtgggct gaagattgtc ctgaggttag
     6181 acatttggtt cacgctgact tcggttccaa caacgttttg actgacaacg gtagaatcac
     6241 tgctgttatc gactggtccg aagctatgtt cggtgactct caatacgagg ttgctaacat
     6301 cttcttttgg agaccatggt tggcttgtat ggaacagcag actagatact tcgagagaag
     6361 acacccagaa ttggctggtt ccccaagatt gagagcttac atgttgagaa tcggtttgga
     6421 ccagttgtac cagtccttgg ttgatggtaa cttcgatgat gctgcttggg ctcaaggtag
     6481 atgtgacgct atcgttagat ccggtgctgg tactgttggt agaactcaga ttgctagaag
     6541 atccgctgct gtttggactg acggttgtgt tgaagttttg gctgactccg gtaacagaag
     6601 accatccact agaccaagag ctaaagaggt tggtagagtt taagtattaa gaacgttatt
     6661 tatatttcaa atttttcttt tttttctcta cagacgctgt acgcatgtaa cattatactg
     6721 aaaaccttgc ttgagaaggt tttgggacgc tcgaagatcc agctgcatta atgaatcggc
     6781 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc ggaattcact
     6841 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     6901 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     6961 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     7021 gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc
     7081 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     7141 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     7201 gaggttttca ccgtcatcac cgaaacgcgc ga
//
