LOCUS       Exported                7544 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 7544)
  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..7544
                     /organism="recombinant plasmid"
                     /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"
     primer_bind     complement(2176..2198)
                     /label=MT1328
                     /note="color: 黒; sequence: 
                     ATCATACATAGGATCCttgttatccgctcacaattccac; added: 2024-08-31"
     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     3880..3899
                     /label=MT2032
                     /note="color: 黒; sequence: caggtcgacTCTAGAGGATC; added: 
                     2024-08-31"
     promoter        join(3901..3930,3933..3962,3965..3994,3997..4026,4029..4058,
                     4061..4090,4112..4141,4144..4173,4176..4205,4208..4237,
                     4240..4269,4272..4301,4308..4474)
                     /label=KpPhl12.1
                     /note="This forward directional feature has 15 segments:
                     1: 3901 .. 3930 / #99ccff / phlO
                     2: 3933 .. 3962 / #99ccff / phlO
                     3: 3965 .. 3994 / #99ccff / phlO
                     4: 3997 .. 4026 / #99ccff / phlO
                     5: 4029 .. 4058 / #99ccff / phlO
                     6: 4061 .. 4090 / #99ccff / phlO
                     7: 4112 .. 4141 / #99ccff / phlO
                     8: 4144 .. 4173 / #99ccff / phlO
                     9: 4176 .. 4205 / #99ccff / phlO
                     10: 4208 .. 4237 / #99ccff / phlO
                     11: 4240 .. 4269 / #99ccff / phlO
                     12: 4272 .. 4301 / #99ccff / phlO
                     13: 4308 .. 4312 / #666699 / KpAOX1 promoter (-167_-163) 
                     14: 4313 .. 4321 / #ff0000 / TATA box
                     15: 4322 .. 4474 / #666699 / KpAOX1 promoter (-153_-1)"
     CDS             4481..5197
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                     1: 4481 .. 4483 / #05fc14
                     2: 4484 .. 4486 / #05fc14 / 1a
                     3: 4487 .. 5197 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      5275..5521
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        5568..5917
                     /label=ScTEF1
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5918..6955
                     /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(7150..7166)
                     /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 gaatgatacg aaacgtaccg tatcgttaag
     3961 gtcaatgata cgaaacgtac cgtatcgtta aggtcaatga tacgaaacgt accgtatcgt
     4021 taaggtgtat gatacgaaac gtaccgtatc gttaaggtag atgatacgaa acgtaccgta
     4081 tcgttaaggt ggcgttgtga tccggagatc catgatacga aacgtaccgt atcgttaagg
     4141 tgaatgatac gaaacgtacc gtatcgttaa ggtcaatgat acgaaacgta ccgtatcgtt
     4201 aaggtcaatg atacgaaacg taccgtatcg ttaaggtgta tgatacgaaa cgtaccgtat
     4261 cgttaaggta gatgatacga aacgtaccgt atcgttaagg tggcgttcag caatatataa
     4321 acagaaggaa gctgccctgt cttaaacctt tttttttatc atcattatta gcttactttc
     4381 ataattgcga ctggttccaa ttgacaagct tttgatttta acgactttta acgacaactt
     4441 gagaagatca aaaaacaact aattattcga aacgactagt atggtttcta agggtgaaga
     4501 gttgttcact ggtgttgttc caatcttggt tgagttggac ggtgacgtta acggacacaa
     4561 gttctctgtt tctggtgaag gtgagggtga cgctacttac ggaaagttga ctttgaagtt
     4621 catctgtact actggtaagt tgccagttcc atggccaact ttggttacta ctttgactta
     4681 cggtgttcag tgtttctcca gatacccaga ccacatgaag cagcacgatt tcttcaagtc
     4741 tgctatgcca gagggttacg ttcaagagag aactatcttc ttcaaggacg acggtaacta
     4801 caagactaga gctgaggtta agttcgaggg tgacacattg gttaacagaa tcgagttgaa
     4861 gggtatcgac ttcaaagagg acggaaacat cttgggtcac aagttggagt acaactacaa
     4921 ctcccacaac gtttacatca tggctgacaa gcagaagaac ggtatcaagg ttaacttcaa
     4981 gatcagacac aacatcgagg acggttccgt tcaattggct gaccactacc aacagaacac
     5041 tccaattggt gacggtccag ttttgttgcc agacaaccac tacttgtcca ctcaatccgc
     5101 tttgtccaag gacccaaacg agaagagaga tcacatggtt ttgttggagt tcgttactgc
     5161 tgctggtatc actttgggta tggacgagtt gtacaagtaa ctcgagcctt agacatgact
     5221 gttcctcagt tcaagttggg cacttacgag aagaccggtc ttgctagatt ctaatcaaga
     5281 ggatgtcaga atgccatttg cctgagagat gcaggcttca tttttgatac ttttttattt
     5341 gtaacctata tagtatagga ttttttttgt cattttgttt cttctcgtac gagcttgctc
     5401 ctgatcagcc tatctcgcag ctgatgaata tcttgtggta ggggtttggg aaaatcattc
     5461 gagtttgatg tttttcttgg tatttcccac tcctcttcag agtacagaag attaagtgag
     5521 acgttcgttt gtgcaagctt agatccccgg gtaccgagct cgaattcgcc gtaccacttc
     5581 aaaacaccca agcacagcat actaaatttc ccctctttct tcctctaggg tgtcgttaat
     5641 tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt ttcttcgtcg
     5701 aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg aaaatttttt tttttgattt
     5761 ttttctcttt cgatgacctc ccattgatat ttaagttaat aaacggtctt caatttctca
     5821 agtttcagtt tcatttttct tgttctatta caactttttt tacttcttgc tcattagaaa
     5881 gaaagcatag caatctaatc taagttttaa ttacaaaatg aagaagccag agttgactgc
     5941 tacttccgtt gagaagttct tgatcgagaa gttcgactcc gtttccgact tgatgcaatt
     6001 gtctgaaggt gaagagtcca gagctttctc ctttgacgtt ggtggtagag gttacgtttt
     6061 gagagttaat tcctgtgctg acggtttcta caaggacaga tacgtttaca gacacttcgc
     6121 ttccgctgct ttgccaattc cagaggtttt ggacattggt gagttctccg agtccttgac
     6181 ttactgtatc tccagaagag ctcagggagt tacattgcaa gacttgccag aaactgagtt
     6241 gccagctgtt ttgcaaccag ttgctgaagc tatggacgct attgctgctg ctgacttgtc
     6301 tcaaacttct ggttttggtc cattcggtcc acagggtatt ggtcagtaca ctacttggag
     6361 agacttcatc tgtgctatcg ctgacccaca tgtttaccac tggcaaactg ttatggacga
     6421 tactgtttcc gcttccgttg ctcaggcttt ggacgaattg atgttgtggg ctgaagattg
     6481 tcctgaggtt agacatttgg ttcacgctga cttcggttcc aacaacgttt tgactgacaa
     6541 cggtagaatc actgctgtta tcgactggtc cgaagctatg ttcggtgact ctcaatacga
     6601 ggttgctaac atcttctttt ggagaccatg gttggcttgt atggaacagc agactagata
     6661 cttcgagaga agacacccag aattggctgg ttccccaaga ttgagagctt acatgttgag
     6721 aatcggtttg gaccagttgt accagtcctt ggttgatggt aacttcgatg atgctgcttg
     6781 ggctcaaggt agatgtgacg ctatcgttag atccggtgct ggtactgttg gtagaactca
     6841 gattgctaga agatccgctg ctgtttggac tgacggttgt gttgaagttt tggctgactc
     6901 cggtaacaga agaccatcca ctagaccaag agctaaagag gttggtagag tttaagtatt
     6961 aagaacgtta tttatatttc aaatttttct tttttttctc tacagacgct gtacgcatgt
     7021 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaagat ccagctgcat
     7081 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     7141 tcggaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     7201 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     7261 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     7321 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     7381 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     7441 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     7501 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcga
//
