LOCUS       sequence-458118-        5584 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For genomic replacement of YOR1 gene in budding yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5584)
  AUTHORS   Gligorovski V, Labagnara M, Rahi SJ
  TITLE     Light-directed evolution of dynamic, multi-state, and computational 
            protein functionalities
  JOURNAL   bioRxiv 2024.02.28.582517
REFERENCE   2  (bases 1 to 5584)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.02.28.582517"
COMMENT     Sequence Label: pVG329
FEATURES             Location/Qualifiers
     source          1..5584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..57)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     157..176
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            complement(255..1830)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(255..452)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(458..1486)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(458..1486)
                     /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(1487..1830)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     rep_origin      complement(1947..2402)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2084..2105)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2296..2315
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2528..2550
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2542..2559
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2543..2559
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2569..2588
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2569..2587
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2612..2628
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2613..2629
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3277..3293)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3277..3293)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3330..3350)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3330..3348)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3369..3385)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3369..3385)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3382..3404)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3393..3409
                     /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)."
     promoter        complement(join(3417..3423,3424..3441,3442..3447))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3462..3483
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3600..3617)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3771..4359)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3851..3870)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4530..5321,5322..5390))
                     /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     5153..5172
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5391..5495)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5563..5581
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
       61 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
      121 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
      181 gagtgcacca taatcatcga tgaattcgag ctcgttttcg acactggatg gcggcgttag
      241 tatcgaatcg acagcagtat agcgaccagc attcacatac gattgacgca tgatattact
      301 ttctgcgcac ttaacttcgc atctgggcag atgatgtcga ggcgaaaaaa aatataaatc
      361 acgctaacat ttgattaaaa tagaacaact acaatataaa aaaactatac aaatgacaag
      421 ttcttgaaaa caagaatctt tttattgtca gtactgatta ttcctttgcc ctcggacgag
      481 tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg gtccagacgg
      541 ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat cggacgattg
      601 cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc aagctctgat
      661 agagttggtc aagaccaatg cggagcatat acgcccggag ccgcggcgat cctgcaagct
      721 ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac cacggcctcc
      781 agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg ctccagtcaa
      841 tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca
      901 cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctcatcg agagcctgcg
      961 cgacggacgc actgacggtg tcgtccatca cagtttgcca gtgatacaca tggggatcag
     1021 caatcgcgca tatgaaatca cgccatgtag tgtattgacc gattccttgc ggtccgaatg
     1081 ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga
     1141 ccggctgcag aacagcgggc agttcggttt caggcaggtc ttgcaacgtg acaccctgtg
     1201 cacggcggga gatgcaatag gtcaggctct cgctgaattc cccaatgtca agcacttccg
     1261 gaatcgggag cgcggccgat gcaaagtgcc gataaacata acgatctttg tagaaaccat
     1321 cggcgcagct atttacccgc aggacatatc cacgccctcc tacatcgaag ctgaaagcac
     1381 gagattcttc gccctccgag agctgcatca ggtcggagac gctgtcgaac ttttcgatca
     1441 gaaacttctc gacagacgtc gcggtgagtt caggcttttt acccatggtt gtttatgttc
     1501 ggatgtgatg tgagaactgt atcctagcaa gattttaaaa ggaagtatat gaaagaagaa
     1561 cctcagtggc aaatcctaac cttttatatt tctctacagg ggcgcggcgt ggggacaatt
     1621 caacgcgtct gtgaggggag cgtttccctg ctcgcaggtc cgcagcgagg agccgtaatt
     1681 tttgcttcgc gccgtgcggc catcaaaatg tatggatgca aatgattata catggggatg
     1741 tatgggctaa atgtacgggc gacagtcaca tcatgcccct gagctgcgca cgtcaagact
     1801 gtcaaggagg gtattctggg cctccatgtc gctggccggg tgacccggcg gggacgaggc
     1861 aagctaaaca gatctggcgc gccttaatta accctatgcg gtgtgaaata ccgcacagat
     1921 gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattcgc
     1981 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2041 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2101 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2161 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2221 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2281 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2341 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2401 gtccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     2461 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     2521 gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca
     2581 ctatagggcg aattgggtac cgggcccccc ctcgaggtcg acggtatcga taagcttaaa
     2641 acttagaagt gaaaatccta aaaccgaagg atatttcaaa tacgtaaaag aagtgaaaga
     2701 taaaataaag cctaaataag gaagaaaaga agggataaca tttttttttg ttactttttg
     2761 cttattcctc acctaaacag aaggaaaaag ccattcttgt ttaaagagta tttttaaagc
     2821 gttcatgaaa agtcacagtc caaggagtat ttcaaattgt cacttgaagt gaaaacaccc
     2881 cagtgcttct cgacatcaga ggtaccagag gtgtttggct tccaatcttc atcaaaggct
     2941 tcaaaaacaa taacgttaac accccaagct ctcatggaac agatatctgc tgtcccttaa
     3001 atgattacca tacatatata tatttattac acatctgtca gaggtagcta gcgaaggtgt
     3061 cactgaaata ttttttgttc cagttagtat aaatacggag gtagaacagc tctccgcgtg
     3121 tatatctttt tttgcgctat acaagaacag gaagaacgca tttccatacc tttttctcct
     3181 tacaggtgcc ctctgagtag tgtcacgaac gaggaaaaag attaatatta ctgtttttat
     3241 attcaaaaag agtaaagccg ttgctatata cgaatggatc cactagttct agagcggccg
     3301 ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     3361 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3421 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3481 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3541 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3601 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3661 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3721 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3781 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3841 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3901 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     3961 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4021 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4081 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4141 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4201 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4261 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4321 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4381 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4441 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4501 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4561 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4621 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4681 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4741 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4801 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4861 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4921 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     4981 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5041 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5101 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5161 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5221 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5281 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5341 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5401 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5461 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5521 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     5581 ccct
//
