LOCUS       sequence-199849-        4893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HygroBR in domestication vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4893)
  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 4893)
  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-HygroBR (01-21)
FEATURES             Location/Qualifiers
     source          1..4893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    196..229
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            284..1859
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        284..627
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             628..1656
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVFDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAT
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      1662..1859
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1922..1955
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2025..2034
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2029..2038
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2068..2087)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2069..2087)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2094..2111)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2094..2110)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2103..2125)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2251..2706
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2338..2357)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2548..2569
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2784..2888
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2889..2957,2958..3749)
                     /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(3107..3126)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3920..4508
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4409..4428
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4662..4679
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4796..4817
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4832..4837,4838..4855,4856..4862)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4870..4886
                     /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)."
     primer_bind     join(4875..4893,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agctatttag gtgacactat agaatactca
       61 agctatgcat ccaacgcgtt gggagctctc ccatatggtc gacctgcagg cggccgcgaa
      121 ttcactagtg attgggcgat gagtggtctc gggagcagct gaagcttcgt acgctgcagg
      181 tcgacaaccc ttaatataac ttcgtataat gtatgctata cgaagttatt aggtctagag
      241 atctgtttag cttgcctcgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa
      301 taccctcctt gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta
      361 catttagccc atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac
      421 ggcgcgaagc aaaaattacg gctcctcgct gcagacctgc gagcagggaa acgctcccct
      481 cacagacgcg ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga
      541 tttgccactg aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc
      601 tcacatcaca tccgaacata aacaaccatg ggtaaaaagc ctgaactcac cgcgacgtct
      661 gtcgagaagt ttctgatcga aaagttcgac agcgtcttcg acctgatgca gctctcggag
      721 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
      781 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
      841 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
      901 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
      961 gttctgcagc cggtcgcgga ggcaatggat gcgatcgctg cggccgatct tagccagacg
     1021 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     1081 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     1141 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     1201 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     1261 ataacagcgg tcattgactg gagcgaggcg acgttcgggg attcccaata cgaggtcgcc
     1321 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     1381 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     1441 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     1501 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     1561 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     1621 cgacgcccca gcactcgtcc gagggcaaag gaataatcag tactgacaat aaaaagattc
     1681 ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt ctattttaat
     1741 caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca gatgcgaagt
     1801 taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc gctatactgc
     1861 tgtcgattcg atactaacgc cgccatccag tgtcgaaaac gagctctcga gaacccttaa
     1921 tataacttcg tataatgtat gctatacgaa gttattaggt gatatcagat ccactagtgg
     1981 cctatgccgc tcgagaccac tcatcgccca atcgaattcc cgcggccgcc atggcggccg
     2041 ggagcatgcg acgtcgggcc caattcgccc tatagtgagt cgtattacaa ttcactggcc
     2101 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     2161 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     2221 caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg
     2281 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     2341 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     2401 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     2461 ttgattaggg tgatggttca cgtagtgggc cagcgccctg atagacggtt tttcgccctt
     2521 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     2581 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     2641 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     2701 caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatcag
     2761 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2821 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2881 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2941 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3001 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3061 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3121 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3181 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3241 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3301 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3361 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3421 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3481 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3541 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3601 gtgggtcgcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3661 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3721 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3781 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3841 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3901 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3961 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4021 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4081 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4141 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4201 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4261 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4321 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4381 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4441 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4501 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4561 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4621 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4681 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     4741 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     4801 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     4861 tgtgtggaat tgtgagcgga taacaatttc aca
//
