LOCUS       Exported                7383 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 7383)
  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..7383
                     /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"
     misc_feature    join(3901..3930,3933..3962,3965..3994,3997..4026,4029..4058,
                     4061..4090,4117..4313)
                     /label=KpPhl6.0
                     /note="This forward directional feature has 9 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: 4117 .. 4151 / #666699 / AOX1 promoter (-197_-163)
                     8: 4152 .. 4160 / #ff0000 / TATA box
                     9: 4161 .. 4313 / #666699 / AOX1 promoter (-153_-1)"
     primer_bind     complement(4097..4126)
                     /label=MT1252
                     /note="color: 黒; sequence: TTAAATTTTGCGCATTATCAtccggatcac; 
                     added: 2024-08-31"
     primer_bind     4107..4145
                     /label=MT1253
                     /note="color: 黒; sequence: 
                     TGATAATGCGCAAAATTTAACTGTTCTAACCCCTACTTG; added: 2024-08-31"
     primer_bind     complement(4312..4334)
                     /label=MT1254
                     /note="color: 黒; sequence: acccttagaaaccatACTAGTCG; added: 
                     2024-08-31"
     CDS             4320..5036
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="This feature has 3 segments:
                     1: 4320 .. 4322 / #05fc14
                     2: 4323 .. 4325 / #05fc14 / 1a
                     3: 4326 .. 5036 / #05fc14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      5114..5360
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        5407..5756
                     /label=ScTEF1
                     /note="color: #ffffff; direction: RIGHT"
     CDS             5757..6794
                     /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(6989..7005)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     primer_bind     complement(7075..7089)
                     /label=MT939
                     /note="color: 黒; sequence: cgCtattacgccagcggatccATG; added:
                     2024-08-31"
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 tccggatgat aatgcgcaaa atttaactgt tctaacccct
     4141 acttgacagc aatatataaa cagaaggaag ctgccctgtc ttaaaccttt ttttttatca
     4201 tcattattag cttactttca taattgcgac tggttccaat tgacaagctt ttgattttaa
     4261 cgacttttaa cgacaacttg agaagatcaa aaaacaacta attattcgaa acgactagta
     4321 tggtttctaa gggtgaagag ttgttcactg gtgttgttcc aatcttggtt gagttggacg
     4381 gtgacgttaa cggacacaag ttctctgttt ctggtgaagg tgagggtgac gctacttacg
     4441 gaaagttgac tttgaagttc atctgtacta ctggtaagtt gccagttcca tggccaactt
     4501 tggttactac tttgacttac ggtgttcagt gtttctccag atacccagac cacatgaagc
     4561 agcacgattt cttcaagtct gctatgccag agggttacgt tcaagagaga actatcttct
     4621 tcaaggacga cggtaactac aagactagag ctgaggttaa gttcgagggt gacacattgg
     4681 ttaacagaat cgagttgaag ggtatcgact tcaaagagga cggaaacatc ttgggtcaca
     4741 agttggagta caactacaac tcccacaacg tttacatcat ggctgacaag cagaagaacg
     4801 gtatcaaggt taacttcaag atcagacaca acatcgagga cggttccgtt caattggctg
     4861 accactacca acagaacact ccaattggtg acggtccagt tttgttgcca gacaaccact
     4921 acttgtccac tcaatccgct ttgtccaagg acccaaacga gaagagagat cacatggttt
     4981 tgttggagtt cgttactgct gctggtatca ctttgggtat ggacgagttg tacaagtaac
     5041 tcgagcctta gacatgactg ttcctcagtt caagttgggc acttacgaga agaccggtct
     5101 tgctagattc taatcaagag gatgtcagaa tgccatttgc ctgagagatg caggcttcat
     5161 ttttgatact tttttatttg taacctatat agtataggat tttttttgtc attttgtttc
     5221 ttctcgtacg agcttgctcc tgatcagcct atctcgcagc tgatgaatat cttgtggtag
     5281 gggtttggga aaatcattcg agtttgatgt ttttcttggt atttcccact cctcttcaga
     5341 gtacagaaga ttaagtgaga cgttcgtttg tgcaagctta gatccccggg taccgagctc
     5401 gaattcgccg taccacttca aaacacccaa gcacagcata ctaaatttcc cctctttctt
     5461 cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa gagaccgcct
     5521 cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc tttttcttga
     5581 aaattttttt ttttgatttt tttctctttc gatgacctcc cattgatatt taagttaata
     5641 aacggtcttc aatttctcaa gtttcagttt catttttctt gttctattac aacttttttt
     5701 acttcttgct cattagaaag aaagcatagc aatctaatct aagttttaat tacaaaatga
     5761 agaagccaga gttgactgct acttccgttg agaagttctt gatcgagaag ttcgactccg
     5821 tttccgactt gatgcaattg tctgaaggtg aagagtccag agctttctcc tttgacgttg
     5881 gtggtagagg ttacgttttg agagttaatt cctgtgctga cggtttctac aaggacagat
     5941 acgtttacag acacttcgct tccgctgctt tgccaattcc agaggttttg gacattggtg
     6001 agttctccga gtccttgact tactgtatct ccagaagagc tcagggagtt acattgcaag
     6061 acttgccaga aactgagttg ccagctgttt tgcaaccagt tgctgaagct atggacgcta
     6121 ttgctgctgc tgacttgtct caaacttctg gttttggtcc attcggtcca cagggtattg
     6181 gtcagtacac tacttggaga gacttcatct gtgctatcgc tgacccacat gtttaccact
     6241 ggcaaactgt tatggacgat actgtttccg cttccgttgc tcaggctttg gacgaattga
     6301 tgttgtgggc tgaagattgt cctgaggtta gacatttggt tcacgctgac ttcggttcca
     6361 acaacgtttt gactgacaac ggtagaatca ctgctgttat cgactggtcc gaagctatgt
     6421 tcggtgactc tcaatacgag gttgctaaca tcttcttttg gagaccatgg ttggcttgta
     6481 tggaacagca gactagatac ttcgagagaa gacacccaga attggctggt tccccaagat
     6541 tgagagctta catgttgaga atcggtttgg accagttgta ccagtccttg gttgatggta
     6601 acttcgatga tgctgcttgg gctcaaggta gatgtgacgc tatcgttaga tccggtgctg
     6661 gtactgttgg tagaactcag attgctagaa gatccgctgc tgtttggact gacggttgtg
     6721 ttgaagtttt ggctgactcc ggtaacagaa gaccatccac tagaccaaga gctaaagagg
     6781 ttggtagagt ttaagtatta agaacgttat ttatatttca aatttttctt ttttttctct
     6841 acagacgctg tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg
     6901 ctcgaagatc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     6961 tgggcgctct tccgcttcct cggaattcac tggccgtcgt tttacaacgt cgtgactggg
     7021 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     7081 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     7141 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
     7201 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     7261 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     7321 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     7381 cga
//
