LOCUS       Exported                7197 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 7197)
  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..7197
                     /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        3901..4127
                     /label=KpPhl1.2
                     /note="This forward directional feature has 6 segments:
                     1: 3901 .. 3905 / #------
                     2: 3906 .. 3935 / #99ccff / phlO
                     3: 3936 .. 3940 / #------
                     4: 3941 .. 3965 / #666699 / AOX1 promoter (-187_-163)
                     5: 3966 .. 3974 / #ff0000 / TATA box
                     6: 3975 .. 4127 / #666699 / AOX1 promoter (-153_-1)"
     primer_bind     3901..3963
                     /label=KN023
                     /note="color: 黒; sequence: 
                     TATGTatgatacgaaacgtaccgtatcgtTAAGGTAGCGTCTGTTCTAACCCCTACTTG
                      ACAG; added: 2024-08-31"
     primer_bind     complement(4126..4148)
                     /label=MT1254
                     /note="color: 黒; sequence: acccttagaaaccatACTAGTCG; added: 
                     2024-08-31"
     CDS             4134..4850
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                     1: 4134 .. 4136 / #05fc14
                     2: 4137 .. 4139 / #05fc14 / 1a
                     3: 4140 .. 4850 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      4928..5174
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        5221..5570
                     /label=ScTEF1
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5571..6608
                     /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(6803..6819)
                     /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 ctgttctaac ccctacttga
     3961 cagcaatata taaacagaag gaagctgccc tgtcttaaac cttttttttt atcatcatta
     4021 ttagcttact ttcataattg cgactggttc caattgacaa gcttttgatt ttaacgactt
     4081 ttaacgacaa cttgagaaga tcaaaaaaca actaattatt cgaaacgact agtatggttt
     4141 ctaagggtga agagttgttc actggtgttg ttccaatctt ggttgagttg gacggtgacg
     4201 ttaacggaca caagttctct gtttctggtg aaggtgaggg tgacgctact tacggaaagt
     4261 tgactttgaa gttcatctgt actactggta agttgccagt tccatggcca actttggtta
     4321 ctactttgac ttacggtgtt cagtgtttct ccagataccc agaccacatg aagcagcacg
     4381 atttcttcaa gtctgctatg ccagagggtt acgttcaaga gagaactatc ttcttcaagg
     4441 acgacggtaa ctacaagact agagctgagg ttaagttcga gggtgacaca ttggttaaca
     4501 gaatcgagtt gaagggtatc gacttcaaag aggacggaaa catcttgggt cacaagttgg
     4561 agtacaacta caactcccac aacgtttaca tcatggctga caagcagaag aacggtatca
     4621 aggttaactt caagatcaga cacaacatcg aggacggttc cgttcaattg gctgaccact
     4681 accaacagaa cactccaatt ggtgacggtc cagttttgtt gccagacaac cactacttgt
     4741 ccactcaatc cgctttgtcc aaggacccaa acgagaagag agatcacatg gttttgttgg
     4801 agttcgttac tgctgctggt atcactttgg gtatggacga gttgtacaag taactcgagc
     4861 cttagacatg actgttcctc agttcaagtt gggcacttac gagaagaccg gtcttgctag
     4921 attctaatca agaggatgtc agaatgccat ttgcctgaga gatgcaggct tcatttttga
     4981 tactttttta tttgtaacct atatagtata ggattttttt tgtcattttg tttcttctcg
     5041 tacgagcttg ctcctgatca gcctatctcg cagctgatga atatcttgtg gtaggggttt
     5101 gggaaaatca ttcgagtttg atgtttttct tggtatttcc cactcctctt cagagtacag
     5161 aagattaagt gagacgttcg tttgtgcaag cttagatccc cgggtaccga gctcgaattc
     5221 gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt tcttcctcta
     5281 gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc
     5341 tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaaattt
     5401 ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt aataaacggt
     5461 cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt ttttacttct
     5521 tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa atgaagaagc
     5581 cagagttgac tgctacttcc gttgagaagt tcttgatcga gaagttcgac tccgtttccg
     5641 acttgatgca attgtctgaa ggtgaagagt ccagagcttt ctcctttgac gttggtggta
     5701 gaggttacgt tttgagagtt aattcctgtg ctgacggttt ctacaaggac agatacgttt
     5761 acagacactt cgcttccgct gctttgccaa ttccagaggt tttggacatt ggtgagttct
     5821 ccgagtcctt gacttactgt atctccagaa gagctcaggg agttacattg caagacttgc
     5881 cagaaactga gttgccagct gttttgcaac cagttgctga agctatggac gctattgctg
     5941 ctgctgactt gtctcaaact tctggttttg gtccattcgg tccacagggt attggtcagt
     6001 acactacttg gagagacttc atctgtgcta tcgctgaccc acatgtttac cactggcaaa
     6061 ctgttatgga cgatactgtt tccgcttccg ttgctcaggc tttggacgaa ttgatgttgt
     6121 gggctgaaga ttgtcctgag gttagacatt tggttcacgc tgacttcggt tccaacaacg
     6181 ttttgactga caacggtaga atcactgctg ttatcgactg gtccgaagct atgttcggtg
     6241 actctcaata cgaggttgct aacatcttct tttggagacc atggttggct tgtatggaac
     6301 agcagactag atacttcgag agaagacacc cagaattggc tggttcccca agattgagag
     6361 cttacatgtt gagaatcggt ttggaccagt tgtaccagtc cttggttgat ggtaacttcg
     6421 atgatgctgc ttgggctcaa ggtagatgtg acgctatcgt tagatccggt gctggtactg
     6481 ttggtagaac tcagattgct agaagatccg ctgctgtttg gactgacggt tgtgttgaag
     6541 ttttggctga ctccggtaac agaagaccat ccactagacc aagagctaaa gaggttggta
     6601 gagtttaagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctctacagac
     6661 gctgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa
     6721 gatccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6781 ctcttccgct tcctcggaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     6841 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     6901 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     6961 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca
     7021 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     7081 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     7141 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga
//
