LOCUS       sequence-382297-        8774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HR donor to integrate UAS1B8TEF(136)-hrGFP-CYC1t into D17 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8774)
  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 8774)
  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_D17_hrGFP
FEATURES             Location/Qualifiers
     source          1..8774
                     /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             2720..3439
                     /codon_start=1
                     /product="humanized Renilla green fluorescent protein"
                     /label=hrGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
                     QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
                     DGGLVEIRSDINLIEEMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
                     QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
                     AQLTSLGKPLGSLHEWV"
     terminator      3447..3694
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3463..3481)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4620..4636)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4620..4636)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4633..4655)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4644..4660
                     /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(4668..4674,4675..4692,4693..4698))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4713..4734
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4851..4868)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5022..5610)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5102..5121)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5781..6572,6573..6641))
                     /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     6404..6423
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6642..6746)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6814..6832
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(join(8770..8774,1..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 cagatagagt
      661 ctcttcaact cgctcaaaaa acaaccgatt gctctcaatc tttggatgag gaatactggc
      721 acccatgact caggtctcac cacttctgtc catcttctaa gtccactata tcacccctcc
      781 aacccctccc cgttccctct cctaccccat atttccgaac ctaattacaa acaaaggtta
      841 aattgagcag ctctaaattc tccacatcaa agtacaaata ccgtgcacag cctccacata
      901 tgcctctgtt cagatcactg cacactctca aacgtctctc aaccatgccc acaatcactc
      961 tcatccacaa ccccaagtgc tccaaatcat gtgctgctct cgagctgctg gagcagaagc
     1021 aaattagccc caatgtcgtg cagtacctcg acacacctct ttccgttgct gagatttcgt
     1081 ctattgttcg gaaactcgac atgaagcccg tcgagctgct cagaaagggc gagccgcaat
     1141 tttctgagct caatttgggg gcggccaaca cgcccgatgc cgagatcatt gatgccatgg
     1201 cgaaacaccc caatctaatc gagcggccta ttctgattgt ggatgacaag gctgtgattg
     1261 gacggcccac agaaaagctc aatgacctgc tgggaaagct gtagatacgc aagtactgag
     1321 taatagaaag agttgatttg gcctaaatat cgtttctaga atttttgaaa aaaagatgcc
     1381 ttcgaccaga tttgaactga tgatctcagc attaagagtg ctgcgcctta ccacttggcc
     1441 acgaaggctg gtttttgaaa agaagaataa ttaaaagtgt gctacgtgaa aattgaaata
     1501 aatatccatg aatcattagc aaaaaaataa ataaaataaa aaatacatca ttagaataaa
     1561 aaaaaaacaa gacatgccct ttatgacctc aaacaccact ccaaggcctg gatgataaac
     1621 atgtttttcc tgtccaagct agtcttctat cactagtggt acccgaattc ctgaggtgtc
     1681 tcacaagtgc cgtgcagtcc cgcccccact tgcttctctt tgtgtgtagt gtacgtacat
     1741 tatcgagacc gttgttcccg cccacctcga tccggtctag actgaggtgt ctcacaagtg
     1801 ccgtgcagtc ccgcccccac ttgcttctct ttgtgtgtag tgtacgtaca ttatcgagac
     1861 cgttgttccc gcccacctcg atccggggat ccctgaggtg tctcacaagt gccgtgcagt
     1921 cccgccccca cttgcttctc tttgtgtgta gtgtacgtac attatcgaga ccgttgttcc
     1981 cgcccacctc gatccgggtc gacctgaggt gtctcacaag tgccgtgcag tcccgccccc
     2041 acttgcttct ctttgtgtgt agtgtacgta cattatcgag accgttgttc ccgcccacct
     2101 cgatccggga gctcctgagg tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc
     2161 tctttgtgtg tagtgtacgt acattatcga gaccgttgtt cccgcccacc tcgatccggt
     2221 ctagactgag gtgtctcaca agtgccgtgc agtcccgccc ccacttgctt ctctttgtgt
     2281 gtagtgtacg tacattatcg agaccgttgt tcccgcccac ctcgatccgg ggatccctga
     2341 ggtgtctcac aagtgccgtg cagtcccgcc cccacttgct tctctttgtg tgtagtgtac
     2401 gtacattatc gagaccgttg ttcccgccca cctcgatccg ggtcgacctg aggtgtctca
     2461 caagtgccgt gcagtcccgc ccccacttgc ttctctttgt gtgtagtgta cgtacattat
     2521 cgagaccgtt gttcccgccc acctcgatcc gggcatgcct gcagcctaga agcttttgtg
     2581 gttgggactt tagccaaggg tataaaagac caccgtcccc gaattacctt tcctcttctt
     2641 ttctctctct ccttgtcaac tcacacccga aatcgttaag catttccttc tgagtataag
     2701 aatcattcaa aggcgcgcca tggtgagcaa gcagatcctg aagaacaccg gcctgcagga
     2761 gatcatgagc ttcaaggtga acctggaggg cgtggtgaac aaccacgtgt tcaccatgga
     2821 gggctgcggc aagggcaaca tcctgttcgg caaccagctg gtgcagatcc gcgtgaccaa
     2881 gggcgccccc ctgcccttcg ccttcgacat cctgagcccc gccttccagt acggcaaccg
     2941 caccttcacc aagtaccccg aggacatcag cgacttcttc atccagagct tccccgccgg
     3001 cttcgtgtac gagcgcaccc tgcgctacga ggacggcggc ctggtggaga tccgcagcga
     3061 catcaacctg atcgaggaga tgttcgtgta ccgcgtggag tacaagggcc gcaacttccc
     3121 caacgacggc cccgtgatga agaagaccat caccggcctg cagcccagct tcgaggtggt
     3181 gtacatgaac gacggcgtgc tggtgggcca ggtgatcctg gtgtaccgcc tgaacagcgg
     3241 caagttctac agctgccaca tgcgcaccct gatgaagagc aagggcgtgg tgaaggactt
     3301 ccccgagtac cacttcatcc agcaccgcct ggagaagacc tacgtggagg acggcggctt
     3361 cgtggagcag cacgagaccg ccatcgccca gctgaccagc ctgggcaagc ccctgggcag
     3421 cctgcacgag tgggtgtaag ctagcctcat gtaattagtt atgtcacgct tacattcacg
     3481 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt
     3541 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt
     3601 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga
     3661 gaaggttttg ggacgctcga aggctttaat ttgccctagg tcacctatat tacggaagca
     3721 gtggtactca agctcagaac gtgctgcgac aagatcaacg aatactggct ggcaagaaca
     3781 aggacggcaa tcgcaccttc aacgacgaac tcatatatca gcctctcaga gcgggaggca
     3841 ttggcctttt gaacattgtc gagcaggcaa agagtctccg tgccacgcga gcggctcgtt
     3901 tctttcaggg atgatactgc tgcaagacaa aatgctatgt ttctcttttc cagcgagaca
     3961 gtagaattgg gctatgggct tgaatcccac cctgctacga taatgaacaa gattcagtgg
     4021 ttcgagacgg gaagctggca ctggcgccac ttcctatctg acttcatgga ccgatgtcta
     4081 ggacttgcaa ggctgtcttc tatagctgta ctggcggagc ctccagccga cttgcgccag
     4141 agggcgcagt ggtcaagaga gaggacgaaa gttgacctgt tcgaacgcca gcgtcgtgaa
     4201 aacccgatcg caaccatgtc tcgggagcaa ggtgcatatc tcgagtcgca aatcgatgtc
     4261 cctgtcaagg tggcaaagga cagctggcga gatcgtttca aggccatgaa gaacaagcac
     4321 tggttgggga cactgccatc cgagcttgat ccgaaccacc tacactggct taagacaaat
     4381 cagttcctga gcaagcacta ccgacgagat gataagagcg ctgctacgct acacatgttg
     4441 cggctgagcc gcctcccctt cacccggtgg taccctaaat caaaatctgt ccagtacaga
     4501 gacattggat gtagttcttg tcaactcttc aaacagaaga actacctcca cgagcacctt
     4561 ttcgtcaatt gtccagcgtc caaggccatc tgccagacga taagcttggc gtaatcatgg
     4621 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4681 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4741 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4801 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4861 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4921 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4981 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5041 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5101 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5161 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5221 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5281 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5341 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5401 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5461 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5521 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5581 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5641 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5701 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5761 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5821 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5881 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5941 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6001 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6061 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6121 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6181 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6241 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6301 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6361 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6421 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6481 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6541 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6601 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6661 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6721 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     6781 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gcccagatcc
     6841 tctagagtcg aagcggccgc tatgtctgat aaaaggatgt aacataggca agctgctcgt
     6901 gagtgttgag tacgaacctt agatccaaat cacccgcacc cacggatata cttgcttgaa
     6961 tatacagtag tatgcggccg ctgcgacacc atatcatata aaactaacaa tgcagtcgac
     7021 gagtatctgt ctgactcgtc attgccgcct ttggagtacg actccaacta tgagtgtgct
     7081 tggatcactt tgacgataca ttcttcgttg gaggctgtgg gtctgacagc tgcgttttcg
     7141 gcgcggttgg ccgacaacaa tatcagctgc aacgtcattg ctggctttca tcatgatcac
     7201 atttttgtcg gcaaaggcga cgcccagaga gccattgacg ttctttctaa tttggaccga
     7261 tagccgtata gtccagtcta tctataagtt caactaactc gtaactatta ccataacata
     7321 tacttcactg ccccagataa ggttccgata aaaagttctg cagactaaat ttatttcagt
     7381 ctcctcttca ccaccaaaat gccctcctac gaagctcgag ctaacgtcca caagtccgcc
     7441 tttgccgctc gagtgctcaa gctcgtggca gccaagaaaa ccaacctgtg tgcttctctg
     7501 gatgttacca ccaccaagga gctcattgag cttgccgata aggtcggacc ttatgtgtgc
     7561 atgatcaaga cccatatcga catcattgac gacttcacct acgccggcac tgtgctcccc
     7621 ctcaaggaac ttgctcttaa gcacggtttc ttcctgttcg aggacagaaa gttcgcagat
     7681 attggcaaca ctgtcaagca ccagtacaag aacggtgtct accgaatcgc cgagtggtcc
     7741 gatatcacca acgcccacgg tgtacccgga accggaatca ttgctggcct gcgagctggt
     7801 gccgaggaaa ctgtctctga acagaagaag gaggacgtct ctgactacga gaactcccag
     7861 tacaaggagt tcctggtccc ctctcccaac gagaagctgg ccagaggtct gctcatgctg
     7921 gccgagctgt cttgcaaggg ctctctggcc actggcgagt actccaagca gaccattgag
     7981 cttgcccgat ccgaccccga gtttgtggtt ggcttcattg cccagaaccg acctaagggc
     8041 gactctgagg actggcttat tctgaccccc ggggtgggtc ttgacgacaa gggagacgct
     8101 ctcggacagc agtaccgaac tgttgaggat gtcatgtcta ccggaacgga tatcataatt
     8161 gtcggccgag gtctgtacgg ccagaaccga gatcctattg aggaggccaa gcgataccag
     8221 aaggctggct gggaggctta ccagaagatt aactgttaga ggttagacta tggatatgta
     8281 atttaactgt gtatatagag agcgtgcaag tatggagcgc ttgttcagct tgtatgatgg
     8341 tcagacgacc tgtctgatcg agtatgtatg atactgcaca acctgtgtat ccgcatgatc
     8401 tgtccaatgg ggcatgttgt tgtgtttctc gatacggaga tgctgggtac aagtagctaa
     8461 tacgattgaa ctacttatac ttatatgagg cttgaagaaa gctgacttgt gtatgactta
     8521 ttctcaacta catccccagt cacaatacca ccactgcact accactacac caaaaccatg
     8581 atcaaaccac ccatggactt cctggaggca gaagaacttg ttatggaaaa gctcaagaga
     8641 gagaagccaa gatactatca agacatgtgt cgcaacttca aggaggacca agctctgtac
     8701 accgagaaac aggcctttgt cgacgatctg gccctttcgt ctcgcgcgtt tcggtgatga
     8761 cggtgaaaac ctct
//
