LOCUS       Exported                7217 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 7217)
  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..7217
                     /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"
     primer_bind     3895..3927
                     /label=KN028
                     /note="color: 黒; sequence: 
                     ttgcggccgcggatcctatgtatgatacgaaacgtaccgtatc; added: 
                     2024-08-31"
     promoter        join(3901..3935,3941..4147)
                     /label=KpPhl1.4
                     /note="This forward directional feature has 5 segments:
                     1: 3901 .. 3905 / #------
                     2: 3906 .. 3935 / #99ccff / phlO
                     3: 3941 .. 3985 / #666699 / AOX1 promoter (-207_-163)
                     4: 3986 .. 3994 / #ff0000 / TATA box
                     5: 3995 .. 4147 / #666699 / AOX1 promoter (-153_-1)"
     primer_bind     3901..3971
                     /label=KN026
                     /note="color: 黒; sequence: 
                     TATGTatgatacgaaacgtaccgtatcgtTAAGGTAGCGTCGTTCATGATCAAAATTTA
                      ACTGTTCTAACC; added: 2024-08-31"
     regulatory      3986..3994
                     /regulatory_class="TATA_box"
                     /locus_tag="TATA"
                     /label=TATA
                     /note="color: #ff8040"
     primer_bind     complement(4146..4168)
                     /label=MT1254
                     /note="color: 黒; sequence: acccttagaaaccatACTAGTCG; added: 
                     2024-08-31"
     CDS             4154..4870
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                     1: 4154 .. 4156 / #05fc14
                     2: 4157 .. 4159 / #05fc14 / 1a
                     3: 4160 .. 4870 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      4948..5194
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        5241..5590
                     /label=ScTEF1
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5591..6628
                     /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(6823..6839)
                     /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 cgttcatgat caaaatttaa
     3961 ctgttctaac ccctacttga cagcaatata taaacagaag gaagctgccc tgtcttaaac
     4021 cttttttttt atcatcatta ttagcttact ttcataattg cgactggttc caattgacaa
     4081 gcttttgatt ttaacgactt ttaacgacaa cttgagaaga tcaaaaaaca actaattatt
     4141 cgaaacgact agtatggttt ctaagggtga agagttgttc actggtgttg ttccaatctt
     4201 ggttgagttg gacggtgacg ttaacggaca caagttctct gtttctggtg aaggtgaggg
     4261 tgacgctact tacggaaagt tgactttgaa gttcatctgt actactggta agttgccagt
     4321 tccatggcca actttggtta ctactttgac ttacggtgtt cagtgtttct ccagataccc
     4381 agaccacatg aagcagcacg atttcttcaa gtctgctatg ccagagggtt acgttcaaga
     4441 gagaactatc ttcttcaagg acgacggtaa ctacaagact agagctgagg ttaagttcga
     4501 gggtgacaca ttggttaaca gaatcgagtt gaagggtatc gacttcaaag aggacggaaa
     4561 catcttgggt cacaagttgg agtacaacta caactcccac aacgtttaca tcatggctga
     4621 caagcagaag aacggtatca aggttaactt caagatcaga cacaacatcg aggacggttc
     4681 cgttcaattg gctgaccact accaacagaa cactccaatt ggtgacggtc cagttttgtt
     4741 gccagacaac cactacttgt ccactcaatc cgctttgtcc aaggacccaa acgagaagag
     4801 agatcacatg gttttgttgg agttcgttac tgctgctggt atcactttgg gtatggacga
     4861 gttgtacaag taactcgagc cttagacatg actgttcctc agttcaagtt gggcacttac
     4921 gagaagaccg gtcttgctag attctaatca agaggatgtc agaatgccat ttgcctgaga
     4981 gatgcaggct tcatttttga tactttttta tttgtaacct atatagtata ggattttttt
     5041 tgtcattttg tttcttctcg tacgagcttg ctcctgatca gcctatctcg cagctgatga
     5101 atatcttgtg gtaggggttt gggaaaatca ttcgagtttg atgtttttct tggtatttcc
     5161 cactcctctt cagagtacag aagattaagt gagacgttcg tttgtgcaag cttagatccc
     5221 cgggtaccga gctcgaattc gccgtaccac ttcaaaacac ccaagcacag catactaaat
     5281 ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa
     5341 aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg
     5401 tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga
     5461 tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta
     5521 ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt
     5581 taattacaaa atgaagaagc cagagttgac tgctacttcc gttgagaagt tcttgatcga
     5641 gaagttcgac tccgtttccg acttgatgca attgtctgaa ggtgaagagt ccagagcttt
     5701 ctcctttgac gttggtggta gaggttacgt tttgagagtt aattcctgtg ctgacggttt
     5761 ctacaaggac agatacgttt acagacactt cgcttccgct gctttgccaa ttccagaggt
     5821 tttggacatt ggtgagttct ccgagtcctt gacttactgt atctccagaa gagctcaggg
     5881 agttacattg caagacttgc cagaaactga gttgccagct gttttgcaac cagttgctga
     5941 agctatggac gctattgctg ctgctgactt gtctcaaact tctggttttg gtccattcgg
     6001 tccacagggt attggtcagt acactacttg gagagacttc atctgtgcta tcgctgaccc
     6061 acatgtttac cactggcaaa ctgttatgga cgatactgtt tccgcttccg ttgctcaggc
     6121 tttggacgaa ttgatgttgt gggctgaaga ttgtcctgag gttagacatt tggttcacgc
     6181 tgacttcggt tccaacaacg ttttgactga caacggtaga atcactgctg ttatcgactg
     6241 gtccgaagct atgttcggtg actctcaata cgaggttgct aacatcttct tttggagacc
     6301 atggttggct tgtatggaac agcagactag atacttcgag agaagacacc cagaattggc
     6361 tggttcccca agattgagag cttacatgtt gagaatcggt ttggaccagt tgtaccagtc
     6421 cttggttgat ggtaacttcg atgatgctgc ttgggctcaa ggtagatgtg acgctatcgt
     6481 tagatccggt gctggtactg ttggtagaac tcagattgct agaagatccg ctgctgtttg
     6541 gactgacggt tgtgttgaag ttttggctga ctccggtaac agaagaccat ccactagacc
     6601 aagagctaaa gaggttggta gagtttaagt attaagaacg ttatttatat ttcaaatttt
     6661 tctttttttt ctctacagac gctgtacgca tgtaacatta tactgaaaac cttgcttgag
     6721 aaggttttgg gacgctcgaa gatccagctg cattaatgaa tcggccaacg cgcggggaga
     6781 ggcggtttgc gtattgggcg ctcttccgct tcctcggaat tcactggccg tcgttttaca
     6841 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     6901 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     6961 cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     7021 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     7081 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     7141 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     7201 atcaccgaaa cgcgcga
//
