LOCUS       sequence-430347-        7072 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Green Fluorescent Protein - P2A - full length 
            cholesterol 25-hydroxylase-like protein from chinook salmon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7072)
  TITLE     Collet Lab Plasmids 2024
REFERENCE   2  (bases 1 to 7072)
  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"
COMMENT     Sequence Label: pcDNA3.1-Hyg-mEGFP-P2A-otch25hb4
FEATURES             Location/Qualifiers
     source          1..7072
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(927..929,930..932,933..1640)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLKE
                     RVTAAGITLGMDELYK"
     primer_bind     complement(969..990)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1230..1249)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1653..1709
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(2494..2511)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2500..2724
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2770..3198
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2857..2876)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3067..3088
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3207..3227)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3212..3541
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3392..3527
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3454..3473
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3590..4615
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3590..4615
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4745..4866
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4782..4801)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4836..4855
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4915..4931)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4915..4931)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4928..4950)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4939..4955
                     /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(4963..4969,4970..4987,4988..4993))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5008..5029
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5146..5163)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5317..5905)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5397..5416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6076..6867,6868..6936))
                     /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     6699..6718
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6937..7041)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga ctcgagatgg tgagcaaggg cgagctgttc accggggtgg
      961 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1021 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1081 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1141 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1201 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1261 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1321 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1381 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     1441 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     1501 ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc aaagacccca
     1561 acgagaagcg cgatcacatg gtcctgaagg agcgcgtgac cgccgccggg atcactctcg
     1621 gcatggacga gctgtacaag gatggaagcg gagctactaa cttcagcctg ctgaagcagg
     1681 ctggagacgt ggaggagaac cctggaccta tgcttccaca gagcctatgg gacttcatcc
     1741 tgggatataa tgcctggctc agatcacctt tcttccctgt gcttttctcc ctcagcatct
     1801 acctcacctt ctgcctgccc ttcgtggtgc tggacctcct ctcccccaga ctggcctgga
     1861 tcaggacctt taagatccag cagaagagcc acgtctcctg gaccatgatg tggagctgcc
     1921 tggctcactc actctacaac catgtagtgt tcatcttccc tctcactgtg ctgcactggt
     1981 tctggagacc agccaccttc atgcccgagg cccccggaac gctgcgtctc atctgggacg
     2041 tggtcgcctg cctcctgctg ttcgacttcc aatacttcat ctggcatctg ttgcaccata
     2101 aggtgccctg gctctaccgg acgttccaca aggtgcacca caagcacaca tccaccttcg
     2161 ccctgaccac ggagtactct ggggcctggg aaaccctgtc tctgggattc ttcgctgggg
     2221 tcaaccctct gctgctgggc tgccaccccc tgacagagat gctcttctat gttctgaata
     2281 tctggctttc tgtggaggac cactctggct atgacctgcc ctggtccacg catagactgg
     2341 tgccctttgg gctctacggt ggagctctgc accacgacct gcaccatctg aagttcaagt
     2401 ccaactatgc tccgtacttc acacactggg acagggtttt tgggacattg cacaagcatt
     2461 cagactgaag cttaagttta aaccgctgat cagcctcgac tgtgccttct agttgccagc
     2521 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     2581 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     2641 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     2701 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     2761 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggggtggtg gttacgcgca
     2821 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2881 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2941 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3001 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3061 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3121 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3181 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3241 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3301 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3361 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3421 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3481 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3541 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtga tgaaaaagcc
     3601 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     3661 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     3721 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     3781 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     3841 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     3901 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     3961 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     4021 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     4081 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     4141 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     4201 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     4261 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     4321 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     4381 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     4441 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     4501 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     4561 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     4621 gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4681 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4741 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4801 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4861 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4921 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4981 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5041 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5101 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5161 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5221 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5281 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5341 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5401 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5461 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5521 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5581 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5641 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5701 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5761 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5821 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5881 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5941 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6001 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6061 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6121 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6181 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6241 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6301 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6361 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6421 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6481 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6541 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6601 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6661 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6721 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6781 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6841 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6901 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6961 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7021 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
