LOCUS       Exported                7177 bp ds-DNA     circular SYN 11-SEP-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7177)
  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..7177
                     /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=MT1591
                     /note="color: 黒; sequence:
                     cgacTCTAGAGGATCCTATGTatgatacgaaacg; added: 2024-08-31"
     promoter        3901..4107
                     /label=KpPhl1.6
                     /note="This forward directional feature has 6 segments:
                     1: 3901 .. 3905 / #------
                     2: 3906 .. 3935 / #99ccff / phlO
                     3: 3936 .. 3940 / #------
                     4: 3941 .. 3945 / #666699 / AOX1 promoter (-167_-163)
                     5: 3946 .. 3954 / #ff0000 / TATA box
                     6: 3955 .. 4107 / #666699 / AOX1 promoter (-153_-1)"
     primer_bind     3901..3967
                     /label=MT1932
                     /note="color: 黒; sequence: 
                     TATGTatgatacgaaacgtaccgtatcgtTAAGGTAGCGTCAGCAATATATAAACAGAA
                      GGAAGCTG; added: 2024-08-31"
     primer_bind     complement(4106..4128)
                     /label=MT1254
                     /note="color: 黒; sequence: acccttagaaaccatACTAGTCG; added: 
                     2024-08-31"
     CDS             4114..4830
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                     1: 4114 .. 4116 / #05fc14
                     2: 4117 .. 4119 / #05fc14 / 1a
                     3: 4120 .. 4830 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      4908..5154
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        5201..5550
                     /label=ScTEF1
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5551..6588
                     /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(6783..6799)
                     /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 tatgtatgat acgaaacgta ccgtatcgtt aaggtagcgt cagcaatata taaacagaag
     3961 gaagctgccc tgtcttaaac cttttttttt atcatcatta ttagcttact ttcataattg
     4021 cgactggttc caattgacaa gcttttgatt ttaacgactt ttaacgacaa cttgagaaga
     4081 tcaaaaaaca actaattatt cgaaacgact agtatggttt ctaagggtga agagttgttc
     4141 actggtgttg ttccaatctt ggttgagttg gacggtgacg ttaacggaca caagttctct
     4201 gtttctggtg aaggtgaggg tgacgctact tacggaaagt tgactttgaa gttcatctgt
     4261 actactggta agttgccagt tccatggcca actttggtta ctactttgac ttacggtgtt
     4321 cagtgtttct ccagataccc agaccacatg aagcagcacg atttcttcaa gtctgctatg
     4381 ccagagggtt acgttcaaga gagaactatc ttcttcaagg acgacggtaa ctacaagact
     4441 agagctgagg ttaagttcga gggtgacaca ttggttaaca gaatcgagtt gaagggtatc
     4501 gacttcaaag aggacggaaa catcttgggt cacaagttgg agtacaacta caactcccac
     4561 aacgtttaca tcatggctga caagcagaag aacggtatca aggttaactt caagatcaga
     4621 cacaacatcg aggacggttc cgttcaattg gctgaccact accaacagaa cactccaatt
     4681 ggtgacggtc cagttttgtt gccagacaac cactacttgt ccactcaatc cgctttgtcc
     4741 aaggacccaa acgagaagag agatcacatg gttttgttgg agttcgttac tgctgctggt
     4801 atcactttgg gtatggacga gttgtacaag taactcgagc cttagacatg actgttcctc
     4861 agttcaagtt gggcacttac gagaagaccg gtcttgctag attctaatca agaggatgtc
     4921 agaatgccat ttgcctgaga gatgcaggct tcatttttga tactttttta tttgtaacct
     4981 atatagtata ggattttttt tgtcattttg tttcttctcg tacgagcttg ctcctgatca
     5041 gcctatctcg cagctgatga atatcttgtg gtaggggttt gggaaaatca ttcgagtttg
     5101 atgtttttct tggtatttcc cactcctctt cagagtacag aagattaagt gagacgttcg
     5161 tttgtgcaag cttagatccc cgggtaccga gctcgaattc gccgtaccac ttcaaaacac
     5221 ccaagcacag catactaaat ttcccctctt tcttcctcta gggtgtcgtt aattacccgt
     5281 actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg
     5341 caataaaaat ttttatcacg tttctttttc ttgaaaattt ttttttttga tttttttctc
     5401 tttcgatgac ctcccattga tatttaagtt aataaacggt cttcaatttc tcaagtttca
     5461 gtttcatttt tcttgttcta ttacaacttt ttttacttct tgctcattag aaagaaagca
     5521 tagcaatcta atctaagttt taattacaaa atgaagaagc cagagttgac tgctacttcc
     5581 gttgagaagt tcttgatcga gaagttcgac tccgtttccg acttgatgca attgtctgaa
     5641 ggtgaagagt ccagagcttt ctcctttgac gttggtggta gaggttacgt tttgagagtt
     5701 aattcctgtg ctgacggttt ctacaaggac agatacgttt acagacactt cgcttccgct
     5761 gctttgccaa ttccagaggt tttggacatt ggtgagttct ccgagtcctt gacttactgt
     5821 atctccagaa gagctcaggg agttacattg caagacttgc cagaaactga gttgccagct
     5881 gttttgcaac cagttgctga agctatggac gctattgctg ctgctgactt gtctcaaact
     5941 tctggttttg gtccattcgg tccacagggt attggtcagt acactacttg gagagacttc
     6001 atctgtgcta tcgctgaccc acatgtttac cactggcaaa ctgttatgga cgatactgtt
     6061 tccgcttccg ttgctcaggc tttggacgaa ttgatgttgt gggctgaaga ttgtcctgag
     6121 gttagacatt tggttcacgc tgacttcggt tccaacaacg ttttgactga caacggtaga
     6181 atcactgctg ttatcgactg gtccgaagct atgttcggtg actctcaata cgaggttgct
     6241 aacatcttct tttggagacc atggttggct tgtatggaac agcagactag atacttcgag
     6301 agaagacacc cagaattggc tggttcccca agattgagag cttacatgtt gagaatcggt
     6361 ttggaccagt tgtaccagtc cttggttgat ggtaacttcg atgatgctgc ttgggctcaa
     6421 ggtagatgtg acgctatcgt tagatccggt gctggtactg ttggtagaac tcagattgct
     6481 agaagatccg ctgctgtttg gactgacggt tgtgttgaag ttttggctga ctccggtaac
     6541 agaagaccat ccactagacc aagagctaaa gaggttggta gagtttaagt attaagaacg
     6601 ttatttatat ttcaaatttt tctttttttt ctctacagac gctgtacgca tgtaacatta
     6661 tactgaaaac cttgcttgag aaggttttgg gacgctcgaa gatccagctg cattaatgaa
     6721 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcggaat
     6781 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     6841 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     6901 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     6961 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     7021 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     7081 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     7141 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga
//
