LOCUS       sequence-385112-        7846 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  over expression vector for killifish fshb tagged with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7846)
  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 7846)
  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: NFfshb-t2a-GFP
FEATURES             Location/Qualifiers
     source          1..7846
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     178..197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     437..458
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(510..563)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(989..1013)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(1010..1213)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1039..1059)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1214..1517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    1540..1656
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(1923..1967)
                     /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    2152..2385
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2878..3003
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             3050..3230
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(3231..3457)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(3485..3505)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3485..3503)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3524..3540)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3524..3540)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3537..3559)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3548..3564
                     /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(3572..3578,3579..3596,3597..3602))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3617..3638
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3755..3772)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3926..4514)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4006..4025)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4685..5476,5477..5545))
                     /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     5308..5327
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5546..5650)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5676..6131)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5813..5834)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6025..6044
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6258..6280
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6272..6289
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6273..6289
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6299..6318
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6299..6317
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      6338..6473
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6513..6634
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6524..6543)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6578..6597
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             6706..6939
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     misc_feature    7026..7614
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             7132..7143
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7541..7561
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(join(7630..7846,1..497,498..500,501..503))
                     /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             complement(join(7633..7846,1..497,498..500,501..503))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7675..7696)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
       61 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      121 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      181 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
      241 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
      301 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
      361 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
      421 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
      481 agctcttcgc ccttgctcac cattctagaa gggccgggat tctcttccac gtcaccgcat
      541 gtcagcagac tgcctctgcc ctcacagccg agtacgtgtg gatggaaggt tttgcagaat
      601 gtgctctctg tgttgcacgc catgcagctg cagctcctgg ctacagggta cgtgaccccc
      661 attggacacc catcgatgtg ctccacctca taggaccagt ccccgttaca ggtgttcacc
      721 tcaggctggt caatgttgtt gtcgtagatg ggatccatgt agaaacaccg gccctcacag
      781 actgtggtgt ggatgagtgt ggtgatgccg cagctctcca cgggaatgct gacatttttt
      841 agatggcaga tgaagctgca gccctgcccc acctctgcca gcaccagcaa cgctgccatg
      901 acaaccagtt gcatggtggc gaccggtgga tcctctagag tcggtgtctt ctatggaggt
      961 caaaacagcg tggatggcgt ctccaggcga tctgacggtt cactaaacga gctctgctta
     1021 tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac
     1081 gacattttgg aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg
     1141 ggtggagact tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc
     1201 aaaaccgcat caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa
     1261 agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt
     1321 caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc
     1381 gtaaatactc cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat
     1441 acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta
     1501 ccgtaagtta tgtaacgcgg aactcccaag cttatcgata aaattttgaa ttttgtaatt
     1561 tgtttttgta attctttagt ttgtatgtct gttgctatta tgtctactat tctttcccct
     1621 gcactatacc ccccaatccc cccttttctt ttaaaagtta accgataccg tcgagatccg
     1681 ttcactaatc gaatggatct gtctctgtct ctctctccac cttcttcttc tattccttcg
     1741 ggcctgtcgg gtcccctcgg ggttgggagg tgggtctgaa acgataatgg tgaatatccc
     1801 tgcctaactc tattcactat agaaagtaca gcaaaaacta ttcttaaacc taccaagcct
     1861 cctactatca ttatgaataa ttttatatac cacagccaat ttgttatgtt aaaccaattc
     1921 cacaaacttg cccatttatc taattccaat aattcttgtt cattcttttc ttgctggttt
     1981 tgcgattctt caattaagga gtgtattaag cttgtgtaat tgttaatttc tctgtcccac
     2041 tccatccagg tcgtgtgatt ccaaatctgt tccagagatt tattactcca actagcattc
     2101 caaggcacag cagtggtgca aatgagtttt ccagagcaac cccaaatccc caggagctgt
     2161 tgatccttta ggtatctttc cacagccagg attcttgcct ggagctgctt gatgccccag
     2221 actgtgagtt gcaacagatg ctgttgcgcc tcaatagccc tcagcaaatt gttctgctgc
     2281 tgcactatac cagacaataa ttgtctggcc tgtaccgtca gcgtcattga cgctgcgccc
     2341 atagtgcttc ctgctgctcc caagaaccca aggaacaaag ctcctattcc cactgctctt
     2401 ttttctctct gcaccactct tctctttgcc ttggtgggtg ctactcctaa tggttcaatt
     2461 tttactactt tatatttata taattcactt ctccaattgt ccctcatatc tcctcctcca
     2521 ggtctgaaga tcagcggccg cttgctgtgc ggtggtctta cttttgtttt gctcttcctc
     2581 tatcttgtct aaagcttcct tggtgtcttt tatctctatc ctttgatgca cacaatagag
     2641 ggttgctact gtattatata atgatctaag ttcttctgat cctgtctgaa gggatggttg
     2701 tagctgtccc agtatttgtc tacagccttc tgatgtttct aacaggccag gattaactgc
     2761 gaatcgttct agctccctgc ttgcccatac tatatgtttt aatttatatt ttttctttcc
     2821 ccctggcctt aaccgaattt tttcccatcg cgatctaatt ctcccccgct taatactgac
     2881 gctctcgcac ccatctctct ccttctagcc tccgctagtc aaaatttttg gcgtactcac
     2941 cagtcgccgc ccctcgcctc ttgccgtgcg cgcttcagca agccgagtcc tgcgtcgaga
     3001 gagctcctct ggtttccctt tcgctttcaa gtccctgttc gggcgccact gctagagatt
     3061 ttccacactg actaaaaggg tctgagggat ctctagttac cagagtcaca caacagacgg
     3121 gcacacacta cttgaagcac tcaaggcaag ctttattgag gcttaagcag tgggttccct
     3181 agttagccag agagctccca ggctcagatc tggtctaacc agagagaccc gtttatgtat
     3241 cgagctaggc acttaaatac aatatctctg caatgcggca attcagtggt tcgtccaatc
     3301 catgtcagac ccgtctgttg ccttcctaat aaggcacgat cgtaccacct tacttccacc
     3361 aatcggcatg cacggtgctt tttctctcct tgtaaggcat gttgctaact catcgttacc
     3421 atgttgcaag actacaagag tattgcataa gactacatta agcttgcagc tccagctttt
     3481 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     3541 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgga
     3601 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3661 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3721 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3781 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3841 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3901 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3961 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4021 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4081 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4141 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4201 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4261 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4321 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4381 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4441 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4501 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4561 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4621 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4681 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4741 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4801 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4861 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4921 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4981 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5041 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5101 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5161 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5221 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5281 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5341 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5401 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5461 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5521 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5581 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5641 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     5701 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     5761 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     5821 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     5881 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     5941 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     6001 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     6061 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     6121 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     6181 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     6241 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta
     6301 atacgactca ctatagggcg aattgggtac gtccctaggc ctccaaaaaa gcctcctcac
     6361 tacttctgga atagctcaga ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt
     6421 agtcagccat ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga
     6481 gttaggggcg ggatagctag agccagacat gataagatac attgatgagt ttggacaaac
     6541 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     6601 atttgtaacc attataagct gcaataaaca agttcctctc actctctgat attcatttct
     6661 ttgcaagtta taaatactga ataataagat gacatgaact actactgcta gagattttcc
     6721 acactgacta aaagggtctg agggatctct agttaccaga gtcacacaac agacgggcac
     6781 acactacttg aagcactcaa ggcaagcttt attgaggctt aagcagtggg ttccctagtt
     6841 agccagagag ctcccaggct cagatctggt ctaaccagag agacccagta caagcaaaaa
     6901 gcagatcttg tcttcgttgg gagtgaatta gcccttccag tccccccttt tcttttaaaa
     6961 agtggctaag atctacagct gccttgtaag tcattggtct taaaggtacc gagctcgaat
     7021 tccaggcggg gaggcggccc aaagggagat ccgactcgtc tgagggcgaa ggcgaagacg
     7081 cggaagaggc cgcagagccg gcagcaggcc gcgggaagga aggtccgctg gattgagggc
     7141 cgaagggacg tagcagaagg acgtcccgcg cagaatccag gtggcaacac aggcgagcag
     7201 ccatggaaag gacgtcagct tccccgacaa caccacggaa ttgtcagtgc ccaacagccg
     7261 agcccctgtc cagcagcggg caaggcaggc ggcgatgagt tccgccgtgg caatagggag
     7321 ggggaaagcg aaagtcccgg aaaggagctg acaggtggtg gcaatgcccc aaccagtggg
     7381 ggttgcgtca gcaaacacag tgcacaccac gccacgttgc ctgacaacgg gccacaactc
     7441 ctcataaaga gacagcaacc aggatttata caaggaggag aaaatgaaag ccatacggga
     7501 agcaatagca tgatacaaag gcattaaagc agcgtatcca catagcgtaa aaggagcaac
     7561 atagttaaga ataccagtca atctttcaca aattttgtaa tccagaggtt gattgtcgac
     7621 gcggccgctt tacttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa
     7681 ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactgggt
     7741 gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg
     7801 gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcgg
//
