LOCUS       Exported                7667 bp ds-DNA     circular SYN 13-SEP-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pPink-HC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7667)
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7667
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        2..940
                     /gene="Pichia pastoris AOX1"
                     /note="AOX1 promoter"
                     /note="inducible promoter, regulated by methanol"
     terminator      1017..1266
                     /gene="S. cerevisiae CYC1"
                     /note="CYC1 terminator"
                     /note="transcription terminator for CYC1"
     promoter        1286..1298
                     /gene="Pichia pastoris ADE2"
                     /note="PpADE2 HC promoter"
                     /note="weak 13-bp promoter fragment that requires high-copy
                     integration for efficient expression"
     CDS             1299..2990
                     /codon_start=1
                     /gene="Pichia pastoris ADE2"
                     /product="phosphoribosylaminoimidazole carboxylase from 
                     Pichia pastoris"
                     /note="PpADE2"
                     /note="essential for the biosynthesis of purine 
                     nucleotides, including adenine"
                     /translation="MDSQVIGILGGGQLGRMIVEAASRLNIKTVILDDGFSPAKHINAA
                     QDHIDGSFKDEEAIAKLAAKCDVLTVEIEHVNTDALKRVQDRTGIKIYPLPETIELIKD
                     KYLQKEHLIKHNISVTKSQGIESNEKALLLFGEENGFPYLLKSRTMAYDGRGNFVVESK
                     EDISKALEFLKDRPLYAEKFAPFVKELAVMVVRSLEGEVFSYPTVETVHKDNICHIVYA
                     PARVNDTIQKKAQILAENTVKTFPGAGIFGVEMFLLSDGELLVNEIAPRPHNSGHYTID
                     ACVTSQFEAHVRAITGLPMPLDFTKLSTSNTNAIMLNVLGAEKSHGELEFCRRALETPG
                     ASVYLYGKTTRLARKMGHINIIGSSMLEAEQKLEYILEESTHLPSSTVSADTKPLVGVI
                     MGSDSDLPVISKGCDILKQFGVPFEVTIVSAHRTPQRMTRYAFEAASRGIKAIIAGAGG
                     AAHLPGMVAAMTPLPVIGVPVKGSTLDGVDSLHSIVQMPRGVPVATVAINNATNAALLA
                     IRILGTIDHKWQKEMSKYMNAMETEVLGKASNLESEGYESYLKNRL"
     promoter        3217..3366
                     /gene="Pichia pastoris TRP2"
                     /note="PpTRP2 promoter"
                     /note="promoter for Pichia pastoris TRP2"
     CDS             3367..4983
                     /codon_start=1
                     /gene="Pichia pastoris TRP2"
                     /product="anthranilate synthase from Pichia pastoris"
                     /note="PpTRP2"
                     /note="catalyzes the initial step of tryptophan 
                     biosynthesis"
                     /translation="MSVSDSHLATDYFGTQLTKQIHPSAESFLDIVKNDRQSHSPETVP
                     NLYPIYKFIQSDFLTPHLAYIKLTNSSETPSPSFLLESAKNGDTVDRYTFMGHSPRKII
                     KTGPLEGAEVDPLVLLEKELKGTRQAQLPGIPRLSGGAIGYISYDCIKYFEPKTERKLK
                     DVLQLPEAALMLFDTIVAFDNVYQRFQVIGNVSLSVDDSDEAILEKYYKTREEVEKISK
                     VVFDNKTVPYYEQKDIIQGQTFTSNIGQEGYENHVRKLKEHILKGDIFQAVPSQRVARP
                     TSLHPFNIYRHLRTVNPSPYMFYIDYLDFQVVGASPELLVKSDNNNKIITHPIAGTLPR
                     GKTIEEDDNYAKQLKSSLKDRAEHVMLVDLARNDINRVCEPTSTTVDRLLTVERFSHVM
                     HLVSEVSGTLRPNKTRFDAFRSIFPAGTVSGAPKVRAMQLIGELEGEKRGVYAGAVGHW
                     SYDGKSMDTCIALRTMVVKDGVAYLQAGGGIVYDSDPYDEYIETMNKMRSNNNTILEAE
                     KIWTDRLARDENQSESEENDQ"
     primer_bind     complement(5045..5061)
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5069..5085
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /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)."
     promoter        complement(5093..5123)
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    5138..5159
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5447..6035)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6206..7066)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7067..7171)
                     /gene="bla"
                     /note="AmpR promoter"
     primer_bind     7645..7661
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"