LOCUS       sequence-329892-        8873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HR donor to integrate UAS1B8TEF(136)-hrGFP-CYC1t into AXP locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
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 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_AXP_hrGFP
FEATURES             Location/Qualifiers
     source          1..8873
                     /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(4374..4390)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4374..4390)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4387..4409)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4398..4414
                     /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(4422..4428,4429..4446,4447..4452))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4467..4488
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4605..4622)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4776..5364)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4856..4875)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5535..6326,6327..6395))
                     /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     6158..6177
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6396..6500)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6568..6586
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8524..8546)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8646..8665
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(8859..8873,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(8873,1..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 cggtacgaac ggcaccagct ggttaatgtg gggttgagag tgcggaacct tgttcgaagg
      361 cccattctac tctgtacacg atttgcaacg gccagcgaga tatcaatgag tacggcgtgt
      421 atatgtctca gggagaaaaa aactccttcg gggtacggaa cctcaaacac cactgatctc
      481 ctaacggctc tagaggagag tcctcttgag cgattgagtc actgaggagc agggttccat
      541 caagtcgcag ataaagaaac aagcaaacaa cgatgccgtg tatctcacac ccacctccaa
      601 gacccttgga agaccagcca tgaaccttag cttccaccaa agcctgaacg gcacgaggcg
      661 atcgtggcaa tccacgtgca agtgtaaatc agtgtttaat ctcgaagctt gcatggttta
      721 gcagggggcc gtcctatagt aactgcacga tagcgccctg ccaagcgatg ccacgacggt
      781 gccaagaaac atggaatgtc ctagttttgg agtggtgcgt tgcggattat gttagcgctg
      841 tgtgttactg ggggaataga gatacgctcc ctcgcagctc aaacgtcaag tgcacccata
      901 tactcggtcg agacccattt ctcgtccttg tccaaagttt gaccctggtt aaggcacatc
      961 agggcctatc gtgataaaca agtactgtac atctgtatgt acgtgggggt actgcgaaaa
     1021 ctgataacgt tgaaatacaa tggcttgaaa tcggtgtcgg tctgagtgaa ttcaagcgat
     1081 tgtacggcaa gatatatatt ggagtctctc aataacatat tgcttttgga tcaatggttt
     1141 tgttggcttc tgctggacca aatacacgtc ccttggtgag acaaacaaac acaccacgcc
     1201 gttatcgccc accttatttg ccttctaata ccgcactata ttggaacttt caactcattg
     1261 accatgcatc tcaccagagg caatgactga ggaccaggtc gaagtaacta 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 ctaggaaaag
     3361 tggttgtacc agaaaacaga tccacaagta cctataaatc actttgtgtg tgaggactct
     3421 ggaactgctc acgagtggtt ccaggctact ggtagttcta ttcgagctcc aaagcacatt
     3481 aagttacgct tggtgaacta tagcttctgg tcctctgctt gcaatgaagc tgtgggtgga
     3541 gtaaacggtg ccgcttaata cagggatggt gcgtgagata ggagatttgg agccgtctac
     3601 tctgtcggcc aacgacataa atagaccccc tcagtcacct tagacacagc agaattccac
     3661 cagatcagct tccaattcta aagatgcagt tctctcttgc cacccttacc accctcttgg
     3721 ccttcgttgc cgcagccccc gcaaacaagg gatttgttca tgcccctatc aagaagcagt
     3781 ccctccaggc ggcccagagc aagatcccca actttgcctc ctccggcccc atcactgccg
     3841 agctctacaa tgagctcatg gcttaccagg ttcaaatttc tcttggtggt cagactatct
     3901 ctgccagcat cgataccgga tccgagattc tgtgggtttg ggagaacgac tctattgctt
     3961 gccaggttga ccagcaggac tgcgacactg acggctctta caaccccaag aagagttcca
     4021 cgagcaagga caccggagtg cctttcaaca tcaactatgg caagggacac gctgatggtt
     4081 acctctacac tgataatgct gtcattggtg gagcttccgc tcctggtttc aagtttggtg
     4141 tcaactctgg tgatctcagc tccggcggct tctccatggc aagcccgaca actacgtggt
     4201 catcactggt gatgtcccca actagactat tattatctag catttcttct acaagaagcc
     4261 ttatggcaac tatataattt aattcattaa aaatgttcta tgctaaatgg tgaggcagtg
     4321 attcaattgc aggttcaaca ttgttggatg caattaagct tggcgtaatc atggtcatag
     4381 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4441 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4501 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4561 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4621 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4681 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4741 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4801 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4861 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4921 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4981 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5041 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5101 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5161 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5221 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5281 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5341 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5401 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5461 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5521 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5581 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5641 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5701 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5761 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5821 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5881 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5941 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6001 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6061 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6121 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6181 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6241 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6301 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6361 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6421 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6481 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     6541 attattatca tgacattaac ctataaaaat aggcgtatca cgaggcccag atcctctaga
     6601 gtcgaagcgg ccgctatgtc tgataaaagg atgtaacata ggcaagctgc tcgtgagtgt
     6661 tgagtacgaa ccttagatcc aaatcacccg cacccacgga tatacttgct tgaatataca
     6721 gtagtatgcg gccgctgcga caccatatca tataaaacta acaatgcagt cgacgagtat
     6781 ctgtctgact cgtcattgcc gcctttggag tacgactcca actatgagtg tgcttggatc
     6841 actttgacga tacattcttc gttggaggct gtgggtctga cagctgcgtt ttcggcgcgg
     6901 ttggccgaca acaatatcag ctgcaacgtc attgctggct ttcatcatga tcacattttt
     6961 gtcggcaaag gcgacgccca gagagccatt gacgttcttt ctaatttgga ccgatagccg
     7021 tatagtccag tctatctata agttcaacta actcgtaact attaccataa catatacttc
     7081 actgccccag ataaggttcc gataaaaagt tctgcagact aaatttattt cagtctcctc
     7141 ttcaccacca aaatgccctc ctacgaagct cgagctaacg tccacaagtc cgcctttgcc
     7201 gctcgagtgc tcaagctcgt ggcagccaag aaaaccaacc tgtgtgcttc tctggatgtt
     7261 accaccacca aggagctcat tgagcttgcc gataaggtcg gaccttatgt gtgcatgatc
     7321 aagacccata tcgacatcat tgacgacttc acctacgccg gcactgtgct ccccctcaag
     7381 gaacttgctc ttaagcacgg tttcttcctg ttcgaggaca gaaagttcgc agatattggc
     7441 aacactgtca agcaccagta caagaacggt gtctaccgaa tcgccgagtg gtccgatatc
     7501 accaacgccc acggtgtacc cggaaccgga atcattgctg gcctgcgagc tggtgccgag
     7561 gaaactgtct ctgaacagaa gaaggaggac gtctctgact acgagaactc ccagtacaag
     7621 gagttcctgg tcccctctcc caacgagaag ctggccagag gtctgctcat gctggccgag
     7681 ctgtcttgca agggctctct ggccactggc gagtactcca agcagaccat tgagcttgcc
     7741 cgatccgacc ccgagtttgt ggttggcttc attgcccaga accgacctaa gggcgactct
     7801 gaggactggc ttattctgac ccccggggtg ggtcttgacg acaagggaga cgctctcgga
     7861 cagcagtacc gaactgttga ggatgtcatg tctaccggaa cggatatcat aattgtcggc
     7921 cgaggtctgt acggccagaa ccgagatcct attgaggagg ccaagcgata ccagaaggct
     7981 ggctgggagg cttaccagaa gattaactgt tagaggttag actatggata tgtaatttaa
     8041 ctgtgtatat agagagcgtg caagtatgga gcgcttgttc agcttgtatg atggtcagac
     8101 gacctgtctg atcgagtatg tatgatactg cacaacctgt gtatccgcat gatctgtcca
     8161 atggggcatg ttgttgtgtt tctcgatacg gagatgctgg gtacaagtag ctaatacgat
     8221 tgaactactt atacttatat gaggcttgaa gaaagctgac ttgtgtatga cttattctca
     8281 actacatccc cagtcacaat accaccactg cactaccact acaccaaaac catgatcaaa
     8341 ccacccatgg acttcctgga ggcagaagaa cttgttatgg aaaagctcaa gagagagaag
     8401 ccaagatact atcaagacat gtgtcgcaac ttcaaggagg accaagctct gtacaccgag
     8461 aaacaggcct ttgtcgacga tctggccctt tcgtctcgcg cgtttcggtg atgacggtga
     8521 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     8581 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     8641 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     8701 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     8761 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     8821 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gtt
//
