LOCUS       sequence-471179-        5154 bp    DNA     circular SYN 29-MAY-2025
DEFINITION  Nb Vector Plasmid (under galactose induction), AmpR (E. coli) and 
            TRP1 (yeast), Cen6/ARS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5154)
  TITLE     High-throughput Activity Reprogramming of Protease (HARP)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5154)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 29, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pN4
FEATURES             Location/Qualifiers
     source          1..5154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1002..1666
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    1218..1335
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     1630..1653
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     1677..1696
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1677..1695
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2263..2937)
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     promoter        complement(2938..3218)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     complement(3235..3254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3354..3376
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3414..3432)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    3473..3976
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     CDS             complement(join(4087..4878,4879..4947))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4710..4729
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4948..5052)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtgc ggccgcccct gaattcagct tttgattaag
      721 ccttctagtc caaaaaacac gtttttttgt catttatttc attttcttag aatagtttag
      781 tttattcatt ttatagtcac gaatgtttta tgattctata tagggttgca aacaagcatt
      841 tttcatttta tgttaaaaca atttcaggtt taccttttat tctgcttgtg gtgacgcgtg
      901 tatccgcccg ctcttttggt cacccatgta tagaccatgg tacgcggcac cgacttatac
      961 cgatgatatt agtcagactt tagtgagggt tgaattcgaa ttttcaaaaa ttcttacttt
     1021 ttttttggat ggacgcaaag aagtttaata atcatattac atggcattac caccatatac
     1081 atatccatat acatatccat atctaatctt acttatatgt tgtggaaatg taaagagccc
     1141 cattatctta gcctaaaaaa accttctctt tggaactttc agtaatacgc ttaactgctc
     1201 attgctatat tgaagtacgg attagaagcc gccgagcggg tgacagccct ccgaaggaag
     1261 actctcctcc gtgcgtcctc gtcttcaccg gtcgcgttcc tgaaacgcag atgtgcctcg
     1321 cgccgcactg ctccgaacaa taaagattct acaatactag cttttatggt tatgaagagg
     1381 aaaaattggc agtaacctgg ccccacaaac cttcaaatga acgaatcaaa ttaacaacca
     1441 taggatgata atgcgattag ttttttagcc ttatttctgg ggtaattaat cagcgaagcg
     1501 atgatttttg atctattaac agatatataa atgcaaaaac tgcataacca ctttaactaa
     1561 tactttcaac attttcggtt tgtattactt cttattcaaa tgtaataaaa gtatcaacaa
     1621 aaaattgtta atatacctct atactttaac gtcaaggaga aaaaaccccg gatccgtaat
     1681 acgactcact ataggggcta gcatgcagtt acttcgctgt ttttcaatat tttctgttat
     1741 tgcttcagtt ttagcaagag accctgcagc ttaagactaa gcatgcatgg tctcataagc
     1801 ttggtaccgc ggctagtaat agctcgagat ctgataacaa cagtgtagat gtaacaaaat
     1861 cgactttgtt cccactgtac ttttagctcg tacaaaatac aatatacttt tcatttctcc
     1921 gtaaacaaca tgttttccca tgtaatatcc ttttctattt ttcgttccgt taccaacttt
     1981 acacatactt tatatagcta ttcacttcta tacactaaaa aactaagaca attttaattt
     2041 tgctgcctgc catatttcaa tttgttataa attcctataa tttatcctat tagtagctaa
     2101 aaaaagatga atgtgaatcg aatcctaaga gaattgagct ccaattcact agtgcactgc
     2161 agtacacctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaggca
     2221 agtgcacaaa caatacttaa ataaatacta ctcagtaata acctatttct tagcattttt
     2281 gacgaaattt gctattttgt tagagtcttt tacaccattt gtctccacac ctccgcttac
     2341 atcaacacca ataacgccat ttaatctaag cgcatcacca acattttctg gcgtcagtcc
     2401 accagctaac ataaaatgta agctttcggg gctctcttgc cttccaaccc agtcagaaat
     2461 cgagttccaa tccaaaagtt cacctgtccc acctgcttct gaatcaaaca agggaataaa
     2521 cgaatgaggt ttctgtgaag ctgcactgag tagtatgttg cagtcttttg gaaatacgag
     2581 tcttttaata actggcaaac cgaggaactc ttggtattct tgccacgact catctccatg
     2641 cagttggacg atatcaatgc cgtaatcatt gaccagagcc aaaacatcct ccttaggttg
     2701 attacgaaac acgccaacca agtatttcgg agtgcctgaa ctatttttat atgcttttac
     2761 aagacttgaa attttccttg caataaccgg gtcaattgtt ctctttctat tgggcacaca
     2821 tataataccc agcaagtcag catcagaatc tagagcacat tctgcggcct ctgtgctctg
     2881 caagccgcaa actttcacca atggaccaga actacctgtg aaattaataa cagacatact
     2941 ccaagctgcc tttgtgtgct taatcacgta tactcacgtg ctcaatagtc accaatgccc
     3001 tccctcttgg ccctctcctt ttcttttttc gaccgaatta attcttaatc ggcaaaaaaa
     3061 gaaaagctcc ggatcaagat tgtacgtaag gtgacaagct atttttcaat aaagaatatc
     3121 ttccactact gccatctggc gtcataactg caaagtacac atatattacg atgctgtcta
     3181 ttaaatgctt cctatattat atatatagta atgtcgttta tggtgcactc tcagtacaat
     3241 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     3301 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tgtccgggag
     3361 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagtcgaa agggcctcgt
     3421 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga gtatcacgtg
     3481 ctataaaaat aattataatt taaatttttt aatataaata tataaattaa aaatagaaag
     3541 taaaaaaaga aattaaagaa aaaatagttt ttgttttccg aagatgtaaa agactctagg
     3601 gggatcgcca acaaatacta ccttttatct tgctcttcct gctctcaggt attaatgccg
     3661 aattgtttca tcttgtctgt gtagaaaacc acacacgaaa atcctgtgat tttacatttt
     3721 acttatcgtt aatcgaatgt atatctattt aatctgcttt tcttgtctaa taaatatata
     3781 tgtaaagtac gctttttgtt gaaatttttt aaacctttgt ttattttttt ttcttcattc
     3841 cgtaactctt ctaccttctt tatttacttt ctaaaatcca aatacaaaac ataaaaataa
     3901 ataaacacag agtaaattcc caaattattc catcattaaa agatacgagg cgcgtgtaag
     3961 ttacaggcaa gcgatccgtc ccgagcggcc gcgattatca aaaaggatct tcacctagat
     4021 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4081 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4141 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4201 tggccccagt gctgcaatga taccgcggga cccacgctca ccggctccag atttatcagc
     4261 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4321 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4381 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4441 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4501 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4561 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4621 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4681 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4741 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4801 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4861 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4921 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4981 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5041 aggggttccg cgcacatttc cccgaaaagt gccacctgtc atgaccaaaa tcccttaacg
     5101 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
