LOCUS       Exported                7122 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 7122)
  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..7122
                     /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=MT1956
                     /note="color: 黒; sequence: 
                     gatccatgatacgaaacgtaccgtatcgttaaggtCTAAAGCAGGATGCCTGATATATA
                      AATC; added: 2024-08-31"
     promoter        3901..4052
                     /locus_tag="DAS1 promoter"
                     /label=KpPhl1.13
                     /label=DAS1 promoter
                     /note="This forward directional feature has 4 segments:
                     1: 3901 .. 3930 / #99ccff / phlO
                     2: 3931 .. 3947 / #3366ff / KpDAS1 promoter (-122_-106)
                     3: 3948 .. 3956 / #ff0000 / TATA box
                     4: 3957 .. 4052 / #3366ff / KpDAS1 promoter (-96_-1)"
     primer_bind     complement(4017..4073)
                     /label=MT1461
                     /note="color: 黒; sequence: 
                     acccttagaaaccatactagtTTTGTTCGATTATTCTCCAGATAAAATCAACAATAG; 
                     added: 2024-08-31"
     CDS             4059..4775
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                     1: 4059 .. 4061 / #05fc14
                     2: 4062 .. 4064 / #05fc14 / 1a
                     3: 4065 .. 4775 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      4853..5099
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        5146..5495
                     /label=ScTEF1
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5496..6533
                     /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(6728..6744)
                     /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 ctaaagcagg atgcctgata tataaatccc
     3961 agatgatctg cttttgaaac tattttcagt atcttgattc gtttacttac aaacaactat
     4021 tgttgatttt atctggagaa taatcgaaca aaactagtat ggtttctaag ggtgaagagt
     4081 tgttcactgg tgttgttcca atcttggttg agttggacgg tgacgttaac ggacacaagt
     4141 tctctgtttc tggtgaaggt gagggtgacg ctacttacgg aaagttgact ttgaagttca
     4201 tctgtactac tggtaagttg ccagttccat ggccaacttt ggttactact ttgacttacg
     4261 gtgttcagtg tttctccaga tacccagacc acatgaagca gcacgatttc ttcaagtctg
     4321 ctatgccaga gggttacgtt caagagagaa ctatcttctt caaggacgac ggtaactaca
     4381 agactagagc tgaggttaag ttcgagggtg acacattggt taacagaatc gagttgaagg
     4441 gtatcgactt caaagaggac ggaaacatct tgggtcacaa gttggagtac aactacaact
     4501 cccacaacgt ttacatcatg gctgacaagc agaagaacgg tatcaaggtt aacttcaaga
     4561 tcagacacaa catcgaggac ggttccgttc aattggctga ccactaccaa cagaacactc
     4621 caattggtga cggtccagtt ttgttgccag acaaccacta cttgtccact caatccgctt
     4681 tgtccaagga cccaaacgag aagagagatc acatggtttt gttggagttc gttactgctg
     4741 ctggtatcac tttgggtatg gacgagttgt acaagtaact cgagccttag acatgactgt
     4801 tcctcagttc aagttgggca cttacgagaa gaccggtctt gctagattct aatcaagagg
     4861 atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt
     4921 aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct
     4981 gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga
     5041 gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagac
     5101 gttcgtttgt gcaagcttag atccccgggt accgagctcg aattcgccgt accacttcaa
     5161 aacacccaag cacagcatac taaatttccc ctctttcttc ctctagggtg tcgttaatta
     5221 cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa
     5281 aaaggcaata aaaattttta tcacgtttct ttttcttgaa aatttttttt tttgattttt
     5341 ttctctttcg atgacctccc attgatattt aagttaataa acggtcttca atttctcaag
     5401 tttcagtttc atttttcttg ttctattaca acttttttta cttcttgctc attagaaaga
     5461 aagcatagca atctaatcta agttttaatt acaaaatgaa gaagccagag ttgactgcta
     5521 cttccgttga gaagttcttg atcgagaagt tcgactccgt ttccgacttg atgcaattgt
     5581 ctgaaggtga agagtccaga gctttctcct ttgacgttgg tggtagaggt tacgttttga
     5641 gagttaattc ctgtgctgac ggtttctaca aggacagata cgtttacaga cacttcgctt
     5701 ccgctgcttt gccaattcca gaggttttgg acattggtga gttctccgag tccttgactt
     5761 actgtatctc cagaagagct cagggagtta cattgcaaga cttgccagaa actgagttgc
     5821 cagctgtttt gcaaccagtt gctgaagcta tggacgctat tgctgctgct gacttgtctc
     5881 aaacttctgg ttttggtcca ttcggtccac agggtattgg tcagtacact acttggagag
     5941 acttcatctg tgctatcgct gacccacatg tttaccactg gcaaactgtt atggacgata
     6001 ctgtttccgc ttccgttgct caggctttgg acgaattgat gttgtgggct gaagattgtc
     6061 ctgaggttag acatttggtt cacgctgact tcggttccaa caacgttttg actgacaacg
     6121 gtagaatcac tgctgttatc gactggtccg aagctatgtt cggtgactct caatacgagg
     6181 ttgctaacat cttcttttgg agaccatggt tggcttgtat ggaacagcag actagatact
     6241 tcgagagaag acacccagaa ttggctggtt ccccaagatt gagagcttac atgttgagaa
     6301 tcggtttgga ccagttgtac cagtccttgg ttgatggtaa cttcgatgat gctgcttggg
     6361 ctcaaggtag atgtgacgct atcgttagat ccggtgctgg tactgttggt agaactcaga
     6421 ttgctagaag atccgctgct gtttggactg acggttgtgt tgaagttttg gctgactccg
     6481 gtaacagaag accatccact agaccaagag ctaaagaggt tggtagagtt taagtattaa
     6541 gaacgttatt tatatttcaa atttttcttt tttttctcta cagacgctgt acgcatgtaa
     6601 cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaagatcc agctgcatta
     6661 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     6721 ggaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     6781 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     6841 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt
     6901 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     6961 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     7021 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     7081 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc ga
//
