LOCUS Exported 8873 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION HR donor to integrate UAS1B8TEF(136)-hrGFP-CYC1t into A08 locus. ACCESSION . VERSION . KEYWORDS pHR_A08_hrGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8873) 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 8873) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8873 /organism="synthetic DNA construct" /mol_type="other DNA" 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(4719..4735) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4719..4735) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4732..4754) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4743..4759 /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(4767..4797) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4812..4833 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4950..4967) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5121..5709) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5201..5220) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5880..6740) /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 6503..6522 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6741..6845) /gene="bla" /label=AmpR promoter primer_bind 6913..6931 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(8869..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 ctgcataaaa 661 cccgcataca cgtgcataat acttgcataa cgatagcata taaactgcac gattgctgca 721 cttgacttta tatcaaactc aatatcagca ccacccgatt ctctgattat ttcatcaact 781 ataggagaga atgagttggt gataagacat cggtaccaat aaaaatcacg tgactattcc 841 acaggccttc tgttatatca tcagatgcct ttgaacattc tcttgtcgaa ctgagtccaa 901 tttcaacaac tctttccaca gtgcagtgcc cggttaggcc aattagggtt tggcaaaaat 961 cggcaatata agacagcaaa ctaaaattta tataaagccc atcgctgcct tgttggagcc 1021 tcgttttttc ccacaacaca agcttagcag taattgggtc ttctattctc aattgcacgc 1081 tccccccctc tccttacaat tcgctcactc tcccgactat ttaacaacgc acgacaaaca 1141 tagcttcaat tccccattat gattatctgg ttatcggtgg aggctaagat ggtgtagctt 1201 ctgcttgtcg agccgtctcg tacggcgcca agacgctgct ggtcgagttc aaggcgctgg 1261 gaggcacctg cgtcaacgtg ggctgtgccc aaaaagatca cgtggaacgc ctccgatctg 1321 gcgggccgaa tccgacaggc ccaggagtac ggcttctccg acgtggaccc caagtccgcc 1381 gacaactttg actggaccgg attcaaggcc aaacgagacg cctacgtcaa acgactcaat 1441 ggcatctacg aacgaaacct ccagaaggag ggcgtggagt acgtgtttgg ttgggccacc 1501 ctctacaagc aggagggtca ggagttcccc ctggtactgg ccaagagcga cgatggcaac 1561 gccaagttgt actccgcaaa gaagattaag attgccaccg gcggcaagcc cattctgccc 1621 gacgtgcctg gagccgagta cggcattgat tactagtggt 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 cttctttgct ctcgagaccc 3721 agcctaagcg agtggcggtg gttggaggag gctacattga cgtggagctg gccggtgtct 3781 tccacggact caactccgaa accacccttt tctgtcgagg ccagacggtg ctccgagcgt 3841 tcgacatcat gatccaggac accatcaccg actactaaca agaagggcat caacgtgctc 3901 aagggctctg gcgtcaagaa gattgtcaag aaggataatg gcgagctcac ctacgggcag 3961 gatggcgccg agaatgatat cactctcgat tcgctcattt ggatattgga cgagagcctc 4021 tcaaggacac cctcaacctg agcgagtttg gcatcaagac caacaagcgg ggctacattg 4081 aggtcaacga gtgccagcaa tcgtctttcg acaacattta ttcgcttgga gacgtttgct 4141 gcaaggtcga gctgacccca tggctattgc tactggacga aatctgtcca accggctgtt 4201 tggtcccgct gagttcaagg atcagaagca ggactacacc gatgttcctt cggccgtatt 4261 ttcccacccc gaggttggct ccaacggcat cacggaggct gccgccaagg agcagtatgg 4321 tgaggacaac gtcaagatct acacctctaa gtttgtcgcc atgtactacg ccatgttgca 4381 ggagaaagcc cctaccgcct acaagctggt gtgtgtcgga aaggacgaga aggttgttgg 4441 tctgcacatt gttggcaccg actcttccga gattctgcag ggttttggcg tggccgttcg 4501 aatgggagct accaaggccg atttcgacaa tgttgtggct ctttgtccca cttctgccga 4561 ggagctggtg acaatgggat ataccgggag cgcggcgaga gatttctctg gtttgggcta 4621 tcgatattta atcaactaga ctccactgtt gctgtcacag ggtcagagaa gatactagag 4681 tacggaagtg cgttccgttg aagcttggcg taatcatggt catagctgtt tcctgtgtga 4741 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 4801 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 4861 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 4921 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 4981 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 5041 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 5101 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 5161 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 5221 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 5281 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt 5341 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 5401 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 5461 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 5521 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 5581 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 5641 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 5701 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 5761 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 5821 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 5881 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 5941 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 6001 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 6061 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 6121 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 6181 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 6241 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 6301 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 6361 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 6421 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 6481 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 6541 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 6601 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 6661 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 6721 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 6781 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 6841 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 6901 ttaacctata aaaataggcg tatcacgagg cccagatcct ctagagtcga agcggccgct 6961 atgtctgata aaaggatgta acataggcaa gctgctcgtg agtgttgagt acgaacctta 7021 gatccaaatc acccgcaccc acggatatac ttgcttgaat atacagtagt atgcggccgc 7081 ttcgacacca tatcatataa aactaacaat gcagtcgacg agtatctgtc tgactcgtca 7141 ttgccgcctt tggagtacga ctccaactat gagtgtgctt ggatcacttt gacgatacat 7201 tcttcgttgg aggctgtggg tctgacagct gcgttttcgg cgcggttggc cgacaacaat 7261 atcagctgca acgtcattgc tggctttcat catgatcaca tttttgtcgg caaaggcgac 7321 gcccagagag ccattgacgt tctttctaat ttggaccgat agccgtatag tccagtctat 7381 ctataagttc aactaactcg taactattac cataacatat acttcactgc cccagataag 7441 gttccgataa aaagttctgc agactaaatt tatttcagtc tcctcttcac caccaaaatg 7501 ccctcctacg aagctcgagc taacgtccac aagtccgcct ttgccgctcg agtgctcaag 7561 ctcgtggcag ccaagaaaac caacctgtgt gcttctctgg atgttaccac caccaaggag 7621 ctcattgagc ttgccgataa ggtcggacct tatgtgtgca tgatcaagac ccatatcgac 7681 atcattgacg acttcaccta cgccggcact gtgctccccc tcaaggaact tgctcttaag 7741 cacggtttct tcctgttcga ggacagaaag ttcgcagata ttggcaacac tgtcaagcac 7801 cagtacaaga acggtgtcta ccgaatcgcc gagtggtccg atatcaccaa cgcccacggt 7861 gtacccggaa ccggaatcat tgctggcctg cgagctggtg ccgaggaaac tgtctctgaa 7921 cagaagaagg aggacgtctc tgactacgag aactcccagt acaaggagtt cctggtcccc 7981 tctcccaacg agaagctggc cagaggtctg ctcatgctgg ccgagctgtc ttgcaagggc 8041 tctctggcca ctggcgagta ctccaagcag accattgagc ttgcccgatc cgaccccgag 8101 tttgtggttg gcttcattgc ccagaaccga cctaagggcg actctgagga ctggcttatt 8161 ctgacccccg gggtgggtct tgacgacaag ggagacgctc tcggacagca gtaccgaact 8221 gttgaggatg tcatgtctac cggaacggat atcataattg tcggccgagg tctgtacggc 8281 cagaaccgag atcctattga ggaggccaag cgataccaga aggctggctg ggaggcttac 8341 cagaagatta actgttagag gttagactat ggatatgtca tttaactgtg tatatagaga 8401 gcgtgcaagt atggagcgct tgttcagctt gtatgatggt cagacgacct gtctgatcga 8461 gtatgtatga tactgcacaa cctgtgtatc cgcatgatct gtccaatggg gcatgttgtt 8521 gtgtttctcg atacggagat gctgggtaca agtagctaat acgattgaac tacttatact 8581 tatatgaggc ttgaagaaag ctgacttgtg tatgacttat tctcaactac atccccagtc 8641 acaataccac cactgcacta ccactacacc aaaaccatga tcaaaccacc catggacttc 8701 ctggaggcag aagaacttgt tatggaaaag ctcaagagag agaagccaag atactatcaa 8761 gacatgtgtc gcaacttcaa ggaggaccaa gctctgtaca ccgagaaaca ggcctttgtc 8821 gacgatctgg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tct //