LOCUS       sequence-244365-        8872 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HR donor to integrate UAS1B8TEF(136)-hrGFP-CYC1t into MFE1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8872)
  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 8872)
  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_MFE1_hrGFP
FEATURES             Location/Qualifiers
     source          1..8872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     331..353
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     345..362
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     346..362
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2719..3438
                     /codon_start=1
                     /product="humanized Renilla green fluorescent protein"
                     /label=hrGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
                     QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
                     DGGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
                     QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
                     AQLTSLGKPLGSLHEWV"
     terminator      3446..3693
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3462..3480)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4718..4734)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4718..4734)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4731..4753)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4742..4758
                     /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(4766..4772,4773..4790,4791..4796))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4811..4832
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4949..4966)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5120..5708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5200..5219)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5879..6670,6671..6739))
                     /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     6502..6521
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6740..6844)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6912..6930
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(join(8868..8872,1..18))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
       61 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
      121 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
      181 taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag
      241 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      301 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      361 gtgaattcga gctcggtacc cgcttttcgt agataatgga atacaaatgg atatccagag
      421 tatacacatg gatagtatac actgacacga caattctgta tctctttatg ttaactactg
      481 tgaggcgtta aatagagctt gatatataaa atgttacatt tcacagtctg aacttttgca
      541 gattacctaa tttggtaaga tattaattat gaactgaaag ttgatggcat ccctaaattt
      601 gatgaaaggg ggatcccccg ggttcgaagg taccaaggaa gcatgcggta ctctacaact
      661 ttgaaatgat gacattttcg agaaaatgag aacaaccgaa tatttgtact ccatattgat
      721 acggaaaaat tcacaatgga atcacacaag atagctctga acaattgagg acccttttct
      781 ccctaaattc attgtggttg cggttgtatt gccaacatgg attacgcccg cccgatgacg
      841 agcgacttgc ttgagccgag gcagagtcta aatcgggaca acgaaaccat aaaccccata
      901 ggaagccaaa gtgaccactg catgacccca gtatataacc tgccccaccc ctccatactt
      961 gctgctttct agatcaagtt ataccggacc tgctaactca attgaggtag aagatacgtt
     1021 attaacacaa tctatttatt caacacacac acacaatgtc tggagaacta agatacgacg
     1081 gaaaggtcgt cattgttacc ggtgccggtg gcggtctcgg taaggcatac gcccttttct
     1141 acggctctcg aggagcctct gttgttgtca acgatcttgg tggcgacttc aagggcgacg
     1201 gtgcccaggc tggcagtggc aagcgagtga gtatcattac aagcgcagcg aagcgaaacg
     1261 acccaaaacg acaccacaca gaaggataaa ctaacaccag gttgccgatg ttgtcgtcga
     1321 cgagattgtt tccaagggag gcaaggctgt tgctaactac gactctgtcg agaacggtga
     1381 caagattgtc gagactgccg tcaaggcttt tggctccgtc cacattgtca tcaacaacgc
     1441 cggtattctc cgagatattt ccttcaagaa gatgaccgac aaggactggg atcttgtcta
     1501 caaggtccac gttttcggtg cctacaaggt tacccgagct gcctggcctt acttccgaaa
     1561 gcagaagtac ggtcgagtta tctctacctc ttccgctgct ggtctttacg gaaacttcgg
     1621 ccagaccaac tactccgctg ccaagctcgc actagtggta cccgaattcc tgaggtgtct
     1681 cacaagtgcc gtgcagtccc gcccccactt gcttctcttt gtgtgtagtg tacgtacatt
     1741 atcgagaccg ttgttcccgc ccacctcgat ccggtctaga ctgaggtgtc tcacaagtgc
     1801 cgtgcagtcc cgcccccact tgcttctctt tgtgtgtagt gtacgtacat tatcgagacc
     1861 gttgttcccg cccacctcga tccggggatc cctgaggtgt ctcacaagtg ccgtgcagtc
     1921 ccgcccccac ttgcttctct ttgtgtgtag tgtacgtaca ttatcgagac cgttgttccc
     1981 gcccacctcg atccgggtcg acctgaggtg tctcacaagt gccgtgcagt cccgccccca
     2041 cttgcttctc tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc cgcccacctc
     2101 gatccgggag ctcctgaggt gtctcacaag tgccgtgcag tcccgccccc acttgcttct
     2161 ctttgtgtgt agtgtacgta cattatcgag accgttgttc ccgcccacct cgatccggtc
     2221 tagactgagg tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc tctttgtgtg
     2281 tagtgtacgt acattatcga gaccgttgtt cccgcccacc tcgatccggg gatccctgag
     2341 gtgtctcaca agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt gtagtgtacg
     2401 tacattatcg agaccgttgt tcccgcccac ctcgatccgg gtcgacctga ggtgtctcac
     2461 aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac gtacattatc
     2521 gagaccgttg ttcccgccca cctcgatccg ggcatgcctg cagcctagaa gcttttgtgg
     2581 ttgggacttt agccaagggt ataaaagacc accgtccccg aattaccttt cctcttcttt
     2641 tctctctctc cttgtcaact cacacccgaa atcgttaagc atttccttct gagtataaga
     2701 atcattcaaa ggcgcgccat ggtgagcaag cagatcctga agaacaccgg cctgcaggag
     2761 atcatgagct tcaaggtgaa cctggagggc gtggtgaaca accacgtgtt caccatggag
     2821 ggctgcggca agggcaacat cctgttcggc aaccagctgg tgcagatccg cgtgaccaag
     2881 ggcgcccccc tgcccttcgc cttcgacatc ctgagccccg ccttccagta cggcaaccgc
     2941 accttcacca agtaccccga ggacatcagc gacttcttca tccagagctt ccccgccggc
     3001 ttcgtgtacg agcgcaccct gcgctacgag gacggcggcc tggtggagat ccgcagcgac
     3061 atcaacctga tcgaggagat gttcgtgtac cgcgtggagt acaagggccg caacttcccc
     3121 aacgacggcc ccgtgatgaa gaagaccatc accggcctgc agcccagctt cgaggtggtg
     3181 tacatgaacg acggcgtgct ggtgggccag gtgatcctgg tgtaccgcct gaacagcggc
     3241 aagttctaca gctgccacat gcgcaccctg atgaagagca agggcgtggt gaaggacttc
     3301 cccgagtacc acttcatcca gcaccgcctg gagaagacct acgtggagga cggcggcttc
     3361 gtggagcagc acgagaccgc catcgcccag ctgaccagcc tgggcaagcc cctgggcagc
     3421 ctgcacgagt gggtgtaagc tagcctcatg taattagtta tgtcacgctt acattcacgc
     3481 cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc
     3541 cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt
     3601 cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag
     3661 aaggttttgg gacgctcgaa ggctttaatt tgccctaggt tcggtgagac tctcgccaag
     3721 gagggtgcca agtacaacat tacttccaac gtcatcgctc ctcttgctgc ttcccgaatg
     3781 accgagacag tcatgcccga ggatatcctc aagctcctca agcctgagta cgttgttcct
     3841 ctggtcggct acctcaccca cgactctgtc accgagtctt atggtattta cgaggtcggt
     3901 gctggttaca tggctaaaat ccgatgggag cgaggcaacg gtgctgtttt caagggcgac
     3961 gacactttca ccccgtctgc tattctgaag cgatgggatg aggtcacctc ttttgagagc
     4021 cccacctacc ctaacggccc tgctgacttc ttcaaatacg ctgaggagtc tgttaagcga
     4081 cccgagaacc cccagggacc taccgtctcc ttcaaggacc aggttgtcat tgtcactgga
     4141 gccggtgctg gcattggccg agcttactct cacctccttg ctaagcttgg tgccaaggtc
     4201 gttgttaacg atttcggtaa ccctcagaag gttgtcgatg aaattaaggc cctcggtggt
     4261 atcgccgtcg ctgacaagaa caacgtcatc cacggtgaga aggttgttca gaccgctatc
     4321 gacgccttcg gtgctgtcca cgccgttgtc aacaacgctg gtattctccg agacaagtct
     4381 ttcgccaaca tggatgatga gatgtggcag ctgatctttg atgtccacct caacggtact
     4441 tactccgtta ccaaggccgc gtggccccac ttccttaagc agaagtacgg ccgtgtcatc
     4501 aacaccacct caacttctgg tatctacggt aacttcggcc aggccaacta ctctgccgcc
     4561 aaggctggta tcctcggttt ctcccgagct cttgctcgag agggtgagaa gtacaacatt
     4621 cttgtcaaca ccattgcccc taacgctggt actgccatga ctgcttctgt cttcactgag
     4681 gagatgctcg agctcttcaa agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4741 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4801 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4861 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4921 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4981 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5041 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5101 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5161 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5221 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5281 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5341 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5401 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5461 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5521 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5581 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5641 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5701 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5761 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5821 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5881 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5941 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6001 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6061 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6121 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6181 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6241 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6301 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6361 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6421 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6481 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6541 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6601 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6661 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6721 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6781 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6841 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6901 taacctataa aaataggcgt atcacgaggc ccagatcctc tagagtcgaa gcggccgcta
     6961 tgtctgataa aaggatgtaa cataggcaag ctgctcgtga gtgttgagta cgaaccttag
     7021 atccaaatca cccgcaccca cggatatact tgcttgaata tacagtagta tgcggccgct
     7081 gcgacaccat atcatataaa actaacaatg cagtcgacga gtatctgtct gactcgtcat
     7141 tgccgccttt ggagtacgac tccaactatg agtgtgcttg gatcactttg acgatacatt
     7201 cttcgttgga ggctgtgggt ctgacagctg cgttttcggc gcggttggcc gacaacaata
     7261 tcagctgcaa cgtcattgct ggctttcatc atgatcacat ttttgtcggc aaaggcgacg
     7321 cccagagagc cattgacgtt ctttctaatt tggaccgata gccgtatagt ccagtctatc
     7381 tataagttca actaactcgt aactattacc ataacatata cttcactgcc ccagataagg
     7441 ttccgataaa aagttctgca gactaaattt atttcagtct cctcttcacc accaaaatgc
     7501 cctcctacga agctcgagct aacgtccaca agtccgcctt tgccgctcga gtgctcaagc
     7561 tcgtggcagc caagaaaacc aacctgtgtg cttctctgga tgttaccacc accaaggagc
     7621 tcattgagct tgccgataag gtcggacctt atgtgtgcat gatcaagacc catatcgaca
     7681 tcattgacga cttcacctac gccggcactg tgctccccct caaggaactt gctcttaagc
     7741 acggtttctt cctgttcgag gacagaaagt tcgcagatat tggcaacact gtcaagcacc
     7801 agtacaagaa cggtgtctac cgaatcgccg agtggtccga tatcaccaac gcccacggtg
     7861 tacccggaac cggaatcatt gctggcctgc gagctggtgc cgaggaaact gtctctgaac
     7921 agaagaagga ggacgtctct gactacgaga actcccagta caaggagttc ctggtcccct
     7981 ctcccaacga gaagctggcc agaggtctgc tcatgctggc cgagctgtct tgcaagggct
     8041 ctctggccac tggcgagtac tccaagcaga ccattgagct tgcccgatcc gaccccgagt
     8101 ttgtggttgg cttcattgcc cagaaccgac ctaagggcga ctctgaggac tggcttattc
     8161 tgacccccgg ggtgggtctt gacgacaagg gagacgctct cggacagcag taccgaactg
     8221 ttgaggatgt catgtctacc ggaacggata tcataattgt cggccgaggt ctgtacggcc
     8281 agaaccgaga tcctattgag gaggccaagc gataccagaa ggctggctgg gaggcttacc
     8341 agaagattaa ctgttagagg ttagactatg gatatgtaat ttaactgtgt atatagagag
     8401 cgtgcaagta tggagcgctt gttcagcttg tatgatggtc agacgacctg tctgatcgag
     8461 tatgtatgat actgcacaac ctgtgtatcc gcatgatctg tccaatgggg catgttgttg
     8521 tgtttctcga tacggagatg ctgggtacaa gtagctaata cgattgaact acttatactt
     8581 atatgaggct tgaagaaagc tgacttgtgt atgacttatt ctcaactaca tccccagtca
     8641 caataccacc actgcactac cactacacca aaaccatgat caaaccaccc atggacttcc
     8701 tggaggcaga agaacttgtt atggaaaagc tcaagagaga gaagccaaga tactatcaag
     8761 acatgtgtcg caacttcaag gaggaccaag ctctgtacac cgagaaacag gcctttgtcg
     8821 acgatctggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ct
//
