LOCUS       sequence-474154-        5858 bp    DNA     circular SYN 30-MAY-2025
DEFINITION  Nb Vector Plasmid (under galactose induction), AmpR (E. coli) and 
            HygroR (yeast), Cen6/ARS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5858)
  TITLE     High-throughput Activity Reprogramming of Protease (HARP)
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5858)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pN3
FEATURES             Location/Qualifiers
     source          1..5858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gene            complement(365..1939)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(365..562)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(567..1595)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(567..1595)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     promoter        complement(3121..3562)
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     primer_bind     complement(3127..3150)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    3445..3562
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     rep_origin      complement(3948..4536)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4028..4047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4639..4743
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4744..4812,4813..5604)
                     /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     complement(4962..4981)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     misc_feature    join(5715..5858,1..360)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 aaaaaaaaat aaacaaaggt ttaaaaaatt tcaacaaaaa gcgtacttta catatatatt
       61 tattagacaa gaaaagcaga ttaaatagat atacattcga ttaacgataa gtaaaatgta
      121 aaatcacagg attttcgtgt gtggttttct acacagacaa gatgaaacaa ttcggcatta
      181 atacctgaga gcaggaagag caagataaaa ggtagtattt gttggcgatc cccctagagt
      241 cttttacatc ttcggaaaac aaaaactatt ttttctttaa tttctttttt tactttctat
      301 ttttaattta tatatttata ttaaaaaatt taaattataa ttatttttat agcacgtgat
      361 actccagtat agcgaccagc attcacatac gattgacgca tgatattact ttctgcgcac
      421 ttaacttcgc atctgggcag atgatgtcga ggcgaaaaaa aatataaatc acgctaacat
      481 ttgattaaaa tagaacaact acaatataaa aaaactatac aaatgacaag ttcttgaaaa
      541 caagaatctt tttattgtca gttactttat tcctttgccc tcggacgagt gctggggcgt
      601 cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc cgcgcttctg
      661 cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc gtcgcatcga
      721 ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata gagttggtca
      781 agaccaatgc ggagcatata cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc
      841 cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca gaagaagatg
      901 ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat gaccgctgtt
      961 atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg
     1021 acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc gacggacgca
     1081 ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc aatcgcgcat
     1141 atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg gccgaacccg
     1201 ctcgtctggc taagatcggc cgcagcgatc gcatccatgg cctccgcgac cggttgcaga
     1261 acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc acggcgggag
     1321 atgcaatagg tcaggctctc gctaaattcc ccaatgtcaa gcacttccgg aatcgggagc
     1381 gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc ggcgcagcta
     1441 tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg
     1501 ccctccgaga gctgcatcag gtcggacacg ctgtcgaact tttcgatcag aaacttctcg
     1561 acagacgtcg cggtgagttc aggcttttta cccatttttg tttatgttcg gatgtgatgt
     1621 gagaactgta tcctagcaag attttaaaag gaagtatatg aaagaagaac ctcagtggca
     1681 aatcctaacc ttttatattt ctctacaggg gcgcggcgtg gggacaattc aacgcgtctg
     1741 tgaggggagc gtttccctgc tcgcaggtct ggagcgagga gccgtaattt ttgcttcgcg
     1801 ccgtgcggcc atcaaaatgt atggatgcaa atgattatac atggggatgt atgggctaaa
     1861 tgtacgggcg acagtcacat catgcccctg agctgcgcac gtcaagactg tcaaggaggg
     1921 tattctgggc ctccatgtcg ctggccgggt gacccggcgg ggacgaggca agctaaactg
     1981 tactgcagtg cactagttgg actgttgtga tatgccatat atgcagtggt gttcgccggc
     2041 gctggttccg gctgtcttgc ttagttgtga gtcgccaggc agtcatcggt atgatctgta
     2101 catgattcgt cagtgtgagc accactgacg agcagatttc cgtctcatgc tcagcataca
     2161 tgggtgacca aaagagcggg cggatacacg cgtcaccaca agcagaataa aaggtaaacc
     2221 tgaaattgtt ttaacataaa atgaaaaatg cttgtttgca accctatata gaatcataaa
     2281 acattcgtga ctataaaatg aataaactaa actattctaa gaaaatgaaa taaatgacaa
     2341 aaaaacgtgt tttttggact agaaggctta atcaaaagct ctcgagttag gatccttact
     2401 tcaagaaggt ttcagaagag atggtgtttg gcaacttgta catcatttgt gggatcaagt
     2461 atggcttgat caagttgtag aagatcaagt aagacataga gtcgatgtag atgattggct
     2521 tgttcttgtt gatcttaacg taacagttca attggaactt gtttctcaaa cccttaacca
     2581 agtattcaac ttcttcgaag gtgaaagatt gggtgttcaa aacgatagac ttgttggtag
     2641 agttcttgtt gtagtcccac ttaccaccgt cgtccatgaa ccagtaagcc aaagacattg
     2701 gggtcaagta gttttcaacc aagttgtttg ggatggtttt cttgttgtta acgatgaaca
     2761 agttagccaa cttgttgaaa gcttggtgct tgaaggtttg agcaccccag gtgataacca
     2821 agttacccaa gtggttaact ctttccttct tgtgtggtgg agacaaaacc cattggtcgt
     2881 acaacaaaca aacgtggtcc atgtaagcct tgttcttcca ttcgaattgc atacagtagg
     2941 tcttaccttc gtcgcgagat cggatgtaag cgtcacccaa gatcaaaccg ataccagctt
     3001 cgaattgttc gatgttcaat tcgatcaatt gagacttgta ttccttcaac aacttagagt
     3061 ttggacccaa gttcataact tggttcttct tgatgttctt catagatctt atagtttttt
     3121 ctccttgacg ttaaagtata gaggtatatt aacaattttt tgttgatact tttatgacat
     3181 ttgaataaga agtaatacaa actgaaaatg ttgaaagtat tagttaaagt ggttatgcag
     3241 cttttccatt tatatatctg ttaatagatc aaaaatcatc gcttcgctga ttaattaccc
     3301 cagaaataag gctaaaaaac taatcgcatt atcatcctat ggttgttaat ttgattcgtt
     3361 aatttgaagg tttgtggggc caggttactg ccaatttttc ctcttcataa ccataaaagc
     3421 tagtattgta gaatctttat tgttcggagc agtgcggcgc gaggcacatc tgcgtttcag
     3481 gaacgcgacc ggtgatgacg aggacgcacg gaggagagtc atccgtcgga gggctgtcgc
     3541 ccgctcggcg gcttctaatc cgtacttcaa tatagcaatg agcagttaag cgtattactg
     3601 aaagttccaa agagaaggtt tttttaggct aagataatgg ggcgttcgtc tgcaattatc
     3661 ggccagttca gtgagacggt attgcgacga attgccacgt tgtcgcagtg tctgtactgt
     3721 gggttaatgt ctgtgatcgc cgtcgtgagt tcgtgtcatc cgcgcatgat tcagatggtc
     3781 ctggagatcg ttgcttgtac cggctgtttg tggctctacc atctagatgc gaattcaggg
     3841 gcggccgcac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3901 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3961 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4021 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4081 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4141 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4201 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4261 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4321 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4381 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4441 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4501 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4561 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gacaggtggc
     4621 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4681 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4741 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4801 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4861 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4921 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4981 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5041 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5101 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5161 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5221 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5281 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     5341 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5401 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     5461 tcccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     5521 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5581 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     5641 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatcgc
     5701 ggccgctcgg gacggatcgc ttgcctgtaa cttacacgcg cctcgtatct tttaatgatg
     5761 gaataatttg ggaatttact ctgtgtttat ttatttttat gttttgtatt tggattttag
     5821 aaagtaaata aagaaggtag aagagttacg gaatgaag
//
