LOCUS       sequence-328347-        8674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HR donor to integrate UAS1B8TEF(136)-hrGFP-CYC1t into XPR2 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8674)
  AUTHORS   Schwartz C, Shabbir-Hussain M, Frogue K, Blenner M, Wheeldon I
  TITLE     Standardized markerless gene integration for pathway engineering in 
            Yarrowia lipolytica.
  JOURNAL   ACS Synth Biol. 2016 Dec 19.
  PUBMED    27989123
REFERENCE   2  (bases 1 to 8674)
  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: "ACS Synth 
            Biol. 2016 Dec 19."
COMMENT     Sequence Label: pHR_XPR2_hrGFP
FEATURES             Location/Qualifiers
     source          1..8674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2375..3094
                     /codon_start=1
                     /product="humanized Renilla green fluorescent protein"
                     /label=hrGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
                     QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
                     DGGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
                     QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
                     AQLTSLGKPLGSLHEWV"
     terminator      3102..3349
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3118..3136)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4175..4191)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4175..4191)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4188..4210)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4199..4215
                     /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(4223..4229,4230..4247,4248..4253))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4268..4289
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4406..4423)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4577..5165)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4657..4676)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5336..6127,6128..6196))
                     /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     5959..5978
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6197..6301)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6369..6387
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8325..8347)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8447..8466
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(8660..8674,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(8674,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttcgagctcg gtacccgctt ttcgtagata atggaataca
       61 aatggatatc cagagtatac acatggatag tatacactga cacgacaatt ctgtatctct
      121 ttatgttaac tactgtgagg cgttaaatag agcttgatat ataaaatgtt acatttcaca
      181 gtctgaactt ttgcagatta cctaatttgg taagatatta attatgaact gaaagttgat
      241 ggcatcccta aatttgatga aagggggatc ccccgggttc gaaggtacca aggaagcatg
      301 cggtacgtaa taagagcctc gacctgggca ggggtgagaa cctcgtcatt gatggacagg
      361 tagtaggagg caagaccggc aacgtgggga caggccatgg aggtaccgga gtagaccaaa
      421 gtaccagagt cggactggta agaggcagag atgatatcgg agccgggggc aaagacatca
      481 acacaagttc cgtagttgga tccctgtcca ccggaccaga cggagatctt atcgctagag
      541 tcaatggaac ccacagtgat gataccagag gtggagcctc caatgttacc gggagagtcg
      601 ttacaggcgt ccacggcatc gtttcccgca gcgatggcga caagcagacc ctcctgggta
      661 gctcgagacc atagggcgtc ctgggaagcg gacttgggtc ctcctccaga gaagttcagc
      721 actcctcgag gcagagtgtc tcgcttggag atgtagtcgt tgagagccca ggtgaagccc
      781 tggttgatga cggagagagc tgcggatcgg cctgcaaaca ccttgacggc caccagcttg
      841 gtgttggcgt cgactccgta tgtctttcct cccacggtac ctgcaacgtg agtgccgtga
      901 ccgagaagat cagcgttctg tgtgtcagcg aagttggctc cccagacagc tcggcctccg
      961 aactcggagt gggtggttcg gataccagag tcaacaacgt aagaaacggt ggggtgcttg
     1021 ccaactgtct ctcgaacgta ggcgtagttt ccagtctggg ccttcttatg agagattcta
     1081 gacaggcccc attgagtgac gggagtagtc tggatagctc gcttggcatt agaagaagca
     1141 ggaatctcgg ggagagacac aatggtatcg ggctcgaccg tcagaacaga cgactccttg
     1201 agcttgtcaa cgatagtgga gttgaacttt ccaacaaatc cgagaaagcc agatccatcc
     1261 gtgtagacag gaagatcaag agcagaggtg ataccgttgg acgagtacta gtggtacccg
     1321 aattcctgag gtgtctcaca agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt
     1381 gtagtgtacg tacattatcg agaccgttgt tcccgcccac ctcgatccgg tctagactga
     1441 ggtgtctcac aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac
     1501 gtacattatc gagaccgttg ttcccgccca cctcgatccg gggatccctg aggtgtctca
     1561 caagtgccgt gcagtcccgc ccccacttgc ttctctttgt gtgtagtgta cgtacattat
     1621 cgagaccgtt gttcccgccc acctcgatcc gggtcgacct gaggtgtctc acaagtgccg
     1681 tgcagtcccg cccccacttg cttctctttg tgtgtagtgt acgtacatta tcgagaccgt
     1741 tgttcccgcc cacctcgatc cgggagctcc tgaggtgtct cacaagtgcc gtgcagtccc
     1801 gcccccactt gcttctcttt gtgtgtagtg tacgtacatt atcgagaccg ttgttcccgc
     1861 ccacctcgat ccggtctaga ctgaggtgtc tcacaagtgc cgtgcagtcc cgcccccact
     1921 tgcttctctt tgtgtgtagt gtacgtacat tatcgagacc gttgttcccg cccacctcga
     1981 tccggggatc cctgaggtgt ctcacaagtg ccgtgcagtc ccgcccccac ttgcttctct
     2041 ttgtgtgtag tgtacgtaca ttatcgagac cgttgttccc gcccacctcg atccgggtcg
     2101 acctgaggtg tctcacaagt gccgtgcagt cccgccccca cttgcttctc tttgtgtgta
     2161 gtgtacgtac attatcgaga ccgttgttcc cgcccacctc gatccgggca tgcctgcagc
     2221 ctagaagctt ttgtggttgg gactttagcc aagggtataa aagaccaccg tccccgaatt
     2281 acctttcctc ttcttttctc tctctccttg tcaactcaca cccgaaatcg ttaagcattt
     2341 ccttctgagt ataagaatca ttcaaaggcg cgccatggtg agcaagcaga tcctgaagaa
     2401 caccggcctg caggagatca tgagcttcaa ggtgaacctg gagggcgtgg tgaacaacca
     2461 cgtgttcacc atggagggct gcggcaaggg caacatcctg ttcggcaacc agctggtgca
     2521 gatccgcgtg accaagggcg cccccctgcc cttcgccttc gacatcctga gccccgcctt
     2581 ccagtacggc aaccgcacct tcaccaagta ccccgaggac atcagcgact tcttcatcca
     2641 gagcttcccc gccggcttcg tgtacgagcg caccctgcgc tacgaggacg gcggcctggt
     2701 ggagatccgc agcgacatca acctgatcga ggagatgttc gtgtaccgcg tggagtacaa
     2761 gggccgcaac ttccccaacg acggccccgt gatgaagaag accatcaccg gcctgcagcc
     2821 cagcttcgag gtggtgtaca tgaacgacgg cgtgctggtg ggccaggtga tcctggtgta
     2881 ccgcctgaac agcggcaagt tctacagctg ccacatgcgc accctgatga agagcaaggg
     2941 cgtggtgaag gacttccccg agtaccactt catccagcac cgcctggaga agacctacgt
     3001 ggaggacggc ggcttcgtgg agcagcacga gaccgccatc gcccagctga ccagcctggg
     3061 caagcccctg ggcagcctgc acgagtgggt gtaagctagc ctcatgtaat tagttatgtc
     3121 acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca
     3181 acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt
     3241 tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact
     3301 gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgcc ctaggccaga
     3361 gagtccagct tctggatttc ggaggcgatc tggtcaacag tagcgctact gtcaaagaca
     3421 acgatgaact gatccttctc atcaagatct cgcttgtggt gcttaaactt cttggactgc
     3481 ttagcgacca cggacagaat agatgagtag gcagcggcgg cagggcccgt cgatggggtt
     3541 tatatcgtgt ttctggacaa gatactatcc agaaactgct ctgattgcgt cacgaaacac
     3601 ggctctcgcc aagacgcgcc aagacgatgc cctcattatc ttggtgcaat acaggtggag
     3661 tgtgtgcatt agttggtgcc gtgccttctc acgatatctg acaggggctt gggtttgggt
     3721 tggaagtcgg attccgctga aacatggtgt tgtgcagatg tgagtctctc aaagtcggtt
     3781 catcatactc cattgacatc atggtacagt gcgtacgtaa aagatggcct gactacaagt
     3841 agtgggatct ctctaacatt gtccatcaca tcgtaaatca caccgcctat ccatgaaccc
     3901 tgatgccgat cgtcgaagtt agaccagggc cagtggtggg tagcgtgggg taggctgcca
     3961 ctgccgttgg aaccccttgt gtccctggac tacaacttaa ccaacatgtc agacaaagga
     4021 gagaaaagtg tccgctccac cgaactgacg ccaggaatgt accgtggcgc tcaagaggtc
     4081 atgtgtaatt aaggatacaa gataagctgc gcccttgaat gcaaaaagca cgcagctcat
     4141 atgttaattt ccaatcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4201 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4261 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4321 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4381 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4441 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4501 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4561 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4621 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4681 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4741 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4801 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4861 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4921 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4981 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5041 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5101 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5161 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5221 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5281 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5341 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5401 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5461 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5521 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5581 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5641 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5701 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5761 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5821 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5881 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5941 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6001 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6061 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6121 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6181 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6241 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6301 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     6361 cctataaaaa taggcgtatc acgaggccca gatcctctag agtcgaagcg gccgctatgt
     6421 ctgataaaag gatgtaacat aggcaagctg ctcgtgagtg ttgagtacga accttagatc
     6481 caaatcaccc gcacccacgg atatacttgc ttgaatatac agtagtatgc ggccgctgcg
     6541 acaccatatc atataaaact aacaatgcag tcgacgagta tctgtctgac tcgtcattgc
     6601 cgcctttgga gtacgactcc aactatgagt gtgcttggat cactttgacg atacattctt
     6661 cgttggaggc tgtgggtctg acagctgcgt tttcggcgcg gttggccgac aacaatatca
     6721 gctgcaacgt cattgctggc tttcatcatg atcacatttt tgtcggcaaa ggcgacgccc
     6781 agagagccat tgacgttctt tctaatttgg accgatagcc gtatagtcca gtctatctat
     6841 aagttcaact aactcgtaac tattaccata acatatactt cactgcccca gataaggttc
     6901 cgataaaaag ttctgcagac taaatttatt tcagtctcct cttcaccacc aaaatgccct
     6961 cctacgaagc tcgagctaac gtccacaagt ccgcctttgc cgctcgagtg ctcaagctcg
     7021 tggcagccaa gaaaaccaac ctgtgtgctt ctctggatgt taccaccacc aaggagctca
     7081 ttgagcttgc cgataaggtc ggaccttatg tgtgcatgat caagacccat atcgacatca
     7141 ttgacgactt cacctacgcc ggcactgtgc tccccctcaa ggaacttgct cttaagcacg
     7201 gtttcttcct gttcgaggac agaaagttcg cagatattgg caacactgtc aagcaccagt
     7261 acaagaacgg tgtctaccga atcgccgagt ggtccgatat caccaacgcc cacggtgtac
     7321 ccggaaccgg aatcattgct ggcctgcgag ctggtgccga ggaaactgtc tctgaacaga
     7381 agaaggagga cgtctctgac tacgagaact cccagtacaa ggagttcctg gtcccctctc
     7441 ccaacgagaa gctggccaga ggtctgctca tgctggccga gctgtcttgc aagggctctc
     7501 tggccactgg cgagtactcc aagcagacca ttgagcttgc ccgatccgac cccgagtttg
     7561 tggttggctt cattgcccag aaccgaccta agggcgactc tgaggactgg cttattctga
     7621 cccccggggt gggtcttgac gacaagggag acgctctcgg acagcagtac cgaactgttg
     7681 aggatgtcat gtctaccgga acggatatca taattgtcgg ccgaggtctg tacggccaga
     7741 accgagatcc tattgaggag gccaagcgat accagaaggc tggctgggag gcttaccaga
     7801 agattaactg ttagaggtta gactatggat atgtaattta actgtgtata tagagagcgt
     7861 gcaagtatgg agcgcttgtt cagcttgtat gatggtcaga cgacctgtct gatcgagtat
     7921 gtatgatact gcacaacctg tgtatccgca tgatctgtcc aatggggcat gttgttgtgt
     7981 ttctcgatac ggagatgctg ggtacaagta gctaatacga ttgaactact tatacttata
     8041 tgaggcttga agaaagctga cttgtgtatg acttattctc aactacatcc ccagtcacaa
     8101 taccaccact gcactaccac tacaccaaaa ccatgatcaa accacccatg gacttcctgg
     8161 aggcagaaga acttgttatg gaaaagctca agagagagaa gccaagatac tatcaagaca
     8221 tgtgtcgcaa cttcaaggag gaccaagctc tgtacaccga gaaacaggcc tttgtcgacg
     8281 atctggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     8341 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     8401 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     8461 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     8521 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     8581 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     8641 tgggtaacgc cagggttttc ccagtcacga cgtt
//
