LOCUS       sequence-424235-        7786 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  an overexpression vector of the killifish FSHB fused to GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7786)
  AUTHORS   Moses E, Franek R, Harel I
  TITLE     A scalable and tunable platform for functional interrogation of 
            peptide hormones in fish.
  JOURNAL   eLife. 2023 Oct 24;12:e85960. doi: 10.7554/eLife.85960.
  PUBMED    37872843
REFERENCE   2  (bases 1 to 7786)
  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: "eLife."; 
            date: "2023-10-24"; volume: "12:e85960. doi"; pages: " 
            10.7554/eLife.85960"
COMMENT     Sequence Label: FSHB-GFP fused
FEATURES             Location/Qualifiers
     source          1..7786
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..125
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(126..194,195..986)
                     /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     complement(344..363)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1157..1745
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1646..1665
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1899..1916
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2033..2054
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2069..2074,2075..2092,2093..2099)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2107..2123
                     /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)."
     primer_bind     2112..2134
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2131..2147
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2131..2147
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2166..2186
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2168..2186
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2214..2440
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             2441..2621
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2668..2793
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3286..3519
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3704..3748
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    4015..4131
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        4154..4457
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4458..4661
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4612..4632
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4658..4682
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(5108..5110,5111..5113,5114..5827)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5108..5110,5111..5113,5114..5824)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5153..5174)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5414..5433)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5761..5782
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5843..6431
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5896..5916)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6314..6325)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6518..6751
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6823..6944
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6860..6879)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6914..6933
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6984..7119
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(7139..7158)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7140..7158)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7168..7185)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7168..7184)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7177..7199)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7326..7781
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7413..7432)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7623..7644
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
       61 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
      121 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      181 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      241 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      301 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      361 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      421 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      481 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      541 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      601 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      661 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      721 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      781 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      841 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      901 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      961 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     1021 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1081 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1141 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1201 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1261 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     1321 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     1381 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     1441 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     1501 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     1561 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     1621 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     1681 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     1741 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     1801 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     1861 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     1921 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     1981 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     2041 gttagctcac tcattaggca ccccaggctt tccactttat gcttccggct cgtatgttgt
     2101 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     2161 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc
     2221 ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca
     2281 aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt
     2341 attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg
     2401 cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca
     2461 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     2521 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     2581 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac
     2641 ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg
     2701 cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga
     2761 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg
     2821 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata
     2881 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca
     2941 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa
     3001 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata
     3061 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc
     3121 gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag
     3181 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
     3241 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
     3301 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
     3361 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
     3421 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
     3481 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
     3541 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
     3601 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
     3661 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt
     3721 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
     3781 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
     3841 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
     3901 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag
     3961 agacagagac agatccattc gattagtgaa cggatctcga cggtatcggt taacttttaa
     4021 aagaaaaggg gggattgggg ggtatagtgc aggggaaaga atagtagaca taatagcaac
     4081 agacatacaa actaaagaat tacaaaaaca aattacaaaa ttcaaaattt tatcgataag
     4141 cttgggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     4201 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     4261 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     4321 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     4381 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     4441 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     4501 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     4561 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     4621 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
     4681 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccga ctctagagga
     4741 tccaccggtc gccaccatgc aactggttgt catggcagcg ttgctggtgc tggcagaggt
     4801 gtggcagggc tgcagcttca tctgccatct aaaaaatgtc agcattcccg tggagagctg
     4861 cggcatcacc acactcatcc acaccacagt ctgtgagggc cggtgtttct acatggatcc
     4921 catctacgac aacaacattg accagcctga ggtgaacacc tgtaacgggg actggtccta
     4981 tgaggtggag cacatcgatg ggtgtcctat gggggtcacg taccctgtag ccaggagctg
     5041 cagctgcatg gcgtgcaaca cagagagcac attctgcaaa accttccatc cacacgtact
     5101 cggctgtatg gtgagcaagg gcgaagagct gttcaccggg gtggtgccca tcctggtcga
     5161 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     5221 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     5281 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     5341 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     5401 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     5461 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     5521 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     5581 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     5641 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     5701 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     5761 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     5821 caagtaaagc ggccgcgtcg acaatcaacc tctggattac aaaatttgtg aaagattgac
     5881 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     5941 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     6001 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     6061 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     6121 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     6181 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct
     6241 gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     6301 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     6361 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     6421 cgcctccccg cctggaattc gagctcggta cctttaagac caatgactta caaggcagct
     6481 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     6541 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     6601 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     6661 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     6721 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag
     6781 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     6841 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     6901 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     6961 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     7021 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     7081 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc
     7141 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     7201 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     7261 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     7321 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     7381 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     7441 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     7501 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     7561 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     7621 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     7681 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     7741 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtg
//
