LOCUS       Exported                7325 bp ds-DNA     circular SYN 06-SEP-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7325)
  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..7325
                     /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"
     misc_feature    1988..2060
                     /label=vector repeat
                     /note="color: #a6acb3"
     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     3585..3607
                     /label=MT1834
                     /note="color: 黒; sequence: GGATATGCTGGCTACAGATCTTG; added: 
                     2024-08-31"
     misc_feature    join(3889..3952,3975..3994,4016..4035,4038..4057,4060..4079,
                     4090..4255)
                     /label=KpHsl5.1
                     /note="This feature has 9 segments:
                     1: 3889 .. 3932 / #------
                     2: 3933 .. 3952 / #ffcc00 / luxO(original)
                     3: 3975 .. 3994 / #ffcc00 / luxO(original)
                     4: 4016 .. 4035 / #ffcc00 / luxO(original)
                     5: 4038 .. 4057 / #ffcc00 / luxO(original)
                     6: 4060 .. 4079 / #ffcc00 / luxO(original)
                     7: 4090 .. 4093 / #666699 / KpAOX1 promoter(-166_-163)
                     8: 4094 .. 4102 / #ff0000 / TATA Box
                     9: 4103 .. 4255 / #666699 / KpAOX1 promoter(-153_-1)"
     primer_bind     complement(4072..4109)
                     /label=MT2011
                     /note="color: 黒; sequence: 
                     ccttctgtttataTATTGCTtcacataCgtacctgtac; added: 2024-08-31"
     primer_bind     complement(4077..4120)
                     /label=MT1941
                     /note="color: 黒; sequence: 
                     cagggcagcTTCCTTCTGTTTATAatccggatcacataCgTACC; added: 
                     2024-08-31"
     primer_bind     4077..4109
                     /label=MT2010
                     /note="color: 黒; sequence:
                     ggtacGtatgtgaAGCAATAtataaacagaagg; added: 2024-08-31"
     regulatory      4094..4102
                     /regulatory_class="TATA_box"
                     /locus_tag="TATA"
                     /label=TATA
                     /note="color: #ff8040"
     primer_bind     4097..4120
                     /label=MT1934
                     /note="color: 黒; sequence: tataaacagaaggaagctgccctg; added:
                     2024-08-31"
     primer_bind     complement(4254..4276)
                     /label=MT1254
                     /note="color: 黒; sequence: acccttagaaaccatACTAGTCG; added: 
                     2024-08-31"
     CDS             4262..4978
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                     1: 4262 .. 4264 / #05fc14
                     2: 4265 .. 4267 / #05fc14 / 1a
                     3: 4268 .. 4978 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      5056..5302
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        5349..5698
                     /label=ScTEF1
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5699..6736
                     /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"
     misc_feature    6852..6924
                     /label=vector repeat
                     /note="color: #a6acb3"
     primer_bind     complement(6931..6947)
                     /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 aggtcgacag cgtgtttttt
     3901 attccttaag ttacgcaagg tcgaacataa gtacctgtag gatcgtacag gtttacgcaa
     3961 ggtcgaacat aagtacctgt aggatcgtac aggtcgctat tacgccagcg gatccacctg
     4021 taggatcgta caggtgaacc tgtaggatcg tacaggtcaa cctgtaggat cgtacaggta
     4081 cgtatgtgaa gcaatatata aacagaagga agctgccctg tcttaaacct ttttttttat
     4141 catcattatt agcttacttt cataattgcg actggttcca attgacaagc ttttgatttt
     4201 aacgactttt aacgacaact tgagaagatc aaaaaacaac taattattcg aaacgactag
     4261 tatggtttct aagggtgaag agttgttcac tggtgttgtt ccaatcttgg ttgagttgga
     4321 cggtgacgtt aacggacaca agttctctgt ttctggtgaa ggtgagggtg acgctactta
     4381 cggaaagttg actttgaagt tcatctgtac tactggtaag ttgccagttc catggccaac
     4441 tttggttact actttgactt acggtgttca gtgtttctcc agatacccag accacatgaa
     4501 gcagcacgat ttcttcaagt ctgctatgcc agagggttac gttcaagaga gaactatctt
     4561 cttcaaggac gacggtaact acaagactag agctgaggtt aagttcgagg gtgacacatt
     4621 ggttaacaga atcgagttga agggtatcga cttcaaagag gacggaaaca tcttgggtca
     4681 caagttggag tacaactaca actcccacaa cgtttacatc atggctgaca agcagaagaa
     4741 cggtatcaag gttaacttca agatcagaca caacatcgag gacggttccg ttcaattggc
     4801 tgaccactac caacagaaca ctccaattgg tgacggtcca gttttgttgc cagacaacca
     4861 ctacttgtcc actcaatccg ctttgtccaa ggacccaaac gagaagagag atcacatggt
     4921 tttgttggag ttcgttactg ctgctggtat cactttgggt atggacgagt tgtacaagta
     4981 actcgagcct tagacatgac tgttcctcag ttcaagttgg gcacttacga gaagaccggt
     5041 cttgctagat tctaatcaag aggatgtcag aatgccattt gcctgagaga tgcaggcttc
     5101 atttttgata cttttttatt tgtaacctat atagtatagg attttttttg tcattttgtt
     5161 tcttctcgta cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt
     5221 aggggtttgg gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca
     5281 gagtacagaa gattaagtga gacgttcgtt tgtgcaagct tagatccccg ggtaccgagc
     5341 tcgaattcgc cgtaccactt caaaacaccc aagcacagca tactaaattt cccctctttc
     5401 ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc
     5461 ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt tctttttctt
     5521 gaaaattttt ttttttgatt tttttctctt tcgatgacct cccattgata tttaagttaa
     5581 taaacggtct tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt
     5641 ttacttcttg ctcattagaa agaaagcata gcaatctaat ctaagtttta attacaaaat
     5701 gaagaagcca gagttgactg ctacttccgt tgagaagttc ttgatcgaga agttcgactc
     5761 cgtttccgac ttgatgcaat tgtctgaagg tgaagagtcc agagctttct cctttgacgt
     5821 tggtggtaga ggttacgttt tgagagttaa ttcctgtgct gacggtttct acaaggacag
     5881 atacgtttac agacacttcg cttccgctgc tttgccaatt ccagaggttt tggacattgg
     5941 tgagttctcc gagtccttga cttactgtat ctccagaaga gctcagggag ttacattgca
     6001 agacttgcca gaaactgagt tgccagctgt tttgcaacca gttgctgaag ctatggacgc
     6061 tattgctgct gctgacttgt ctcaaacttc tggttttggt ccattcggtc cacagggtat
     6121 tggtcagtac actacttgga gagacttcat ctgtgctatc gctgacccac atgtttacca
     6181 ctggcaaact gttatggacg atactgtttc cgcttccgtt gctcaggctt tggacgaatt
     6241 gatgttgtgg gctgaagatt gtcctgaggt tagacatttg gttcacgctg acttcggttc
     6301 caacaacgtt ttgactgaca acggtagaat cactgctgtt atcgactggt ccgaagctat
     6361 gttcggtgac tctcaatacg aggttgctaa catcttcttt tggagaccat ggttggcttg
     6421 tatggaacag cagactagat acttcgagag aagacaccca gaattggctg gttccccaag
     6481 attgagagct tacatgttga gaatcggttt ggaccagttg taccagtcct tggttgatgg
     6541 taacttcgat gatgctgctt gggctcaagg tagatgtgac gctatcgtta gatccggtgc
     6601 tggtactgtt ggtagaactc agattgctag aagatccgct gctgtttgga ctgacggttg
     6661 tgttgaagtt ttggctgact ccggtaacag aagaccatcc actagaccaa gagctaaaga
     6721 ggttggtaga gtttaagtat taagaacgtt atttatattt caaatttttc ttttttttct
     6781 ctacagacgc tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga
     6841 cgctcgaaga tccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6901 attgggcgct cttccgcttc ctcggaattc actggccgtc gttttacaac gtcgtgactg
     6961 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     7021 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     7081 cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     7141 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     7201 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     7261 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     7321 cgcga
//
