LOCUS       sequence-199832-        4674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  G418R in domestication vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4674)
  AUTHORS   Perez-Gonzalez A, Kniewel R, Veldhuizen M, Verma HK, 
            Navarro-Rodriguez M, Rubio LM, Caro E
  TITLE     Adaptation of the GoldenBraid modular cloning system and creation of
            a toolkit for the expression of heterologous proteins in yeast 
            mitochondria.
  JOURNAL   BMC Biotechnol. 2017 Nov 13;17(1):80. doi: 
            10.1186/s12896-017-0393-y.
  PUBMED    29132331
REFERENCE   2  (bases 1 to 4674)
  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: "BMC 
            Biotechnol."; date: "2017-11-13"; volume: "17(1):80. doi"; pages: " 
            10.1186/s12896-017-0393-y"
COMMENT     Sequence Label: pUPD-G418R (01-21)
FEATURES             Location/Qualifiers
     source          1..4674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      73..82
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      77..86
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    164..197
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            252..1608
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        252..595
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             596..1405
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRRAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(663..682)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1411..1608
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1671..1704
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        complement(1839..1857)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1840..1857)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1875..1891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1875..1891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1888..1910)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1899..1915
                     /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(1923..1929,1930..1947,1948..1953))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1968..1989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2106..2123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2277..2865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2357..2376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3036..3827,3828..3896))
                     /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     3659..3678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3897..4001)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4079..4534)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4216..4237)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4428..4447
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(4660..4674,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4674,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggcccgacgt
       61 cgcatgctcc cggccgccat ggcggccgcg ggaattcgat tgggcgatga gtggtctcgg
      121 gagcagctga agcttcgtac gctgcaggtc gacaaccctt aatataactt cgtataatgt
      181 atgctatacg aagttattag gtctagagat ctgtttagct tgcctcgtcc ccgccgggtc
      241 acccggccag cgacatggag gcccagaata ccctccttga cagtcttgac gtgcgcagct
      301 caggggcatg atgtgactgt cgcccgtaca tttagcccat acatccccat gtataatcat
      361 ttgcatccat acattttgat ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc
      421 agacctgcga gcagggaaac gctcccctca cagacgcgtt gaattgtccc cacgccgcgc
      481 ccctgtagag aaatataaaa ggttaggatt tgccactgag gttcttcttt catatacttc
      541 cttttaaaat cttgctagga tacagttctc acatcacatc cgaacataaa caaccatggg
      601 taaggaaaag actcacgttt cgaggccgcg attaaattcc aacatggatg ctgatttata
      661 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta
      721 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
      781 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
      841 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggcaa
      901 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
      961 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     1021 tcgcgtattt cgtcgcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     1081 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     1141 gcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     1201 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     1261 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     1321 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     1381 tttgatgctc gatgagtttt tctaatcagt actgacaata aaaagattct tgttttcaag
     1441 aacttgtcat ttgtatagtt tttttatatt gtagttgttc tattttaatc aaatgttagc
     1501 gtgatttata ttttttttcg cctcgacatc atctgcccag atgcgaagtt aagtgcgcag
     1561 aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg ctatactgct gtcgattcga
     1621 tactaacgcc gccatccagt gtcgaaaacg agctctcgag aacccttaat ataacttcgt
     1681 ataatgtatg ctatacgaag ttattaggtg atatcagatc cactagtggc ctatgccgct
     1741 cgagaccact catcgcccaa tcactagtga attcgcggcc gcctgcaggt cgaccatatg
     1801 ggagagctcc caacgcgttg gatgcatagc ttgagtattc tatagtgtca cctaaatagc
     1861 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1921 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     1981 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     2041 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2101 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2161 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2221 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2281 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2341 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2401 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2461 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2521 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2581 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2641 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2701 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2761 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2821 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2881 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2941 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3001 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3061 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3121 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgcgac
     3181 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3241 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3301 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3361 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3421 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3481 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3541 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3601 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3661 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3721 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3781 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3841 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3901 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3961 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4021 ccacctgatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa
     4081 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     4141 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     4201 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     4261 gtcaaagggc gaaaaaccgt ctatcagggc gctggcccac tacgtgaacc atcaccctaa
     4321 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     4381 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     4441 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     4501 cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact
     4561 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     4621 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgtt
//
