LOCUS       sequence-258783-        8932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Prgef-1 INS-2::GFP unc-54 3' UTR C.elegans pan-neural expression of 
            INS-2::GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8932)
  AUTHORS   Hung WL, Wang Y, Chitturi J, Zhen M
  TITLE     A Caenorhabditis elegans developmental decision requires insulin 
            signaling-mediated neuron-intestine communication.
  JOURNAL   Development. 2014 Apr;141(8):1767-79. doi: 10.1242/dev.103846. Epub 
            2014 Mar 26.
  PUBMED    24671950
REFERENCE   2  (bases 1 to 8932)
  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"; doi: 
            "10.1242/dev.103846"; journalName: "Development"; date: "2014-04"; 
            volume: "141"; issue: "8"; pages: "1767-79"
COMMENT     Sequence Label: pJH2947
FEATURES             Location/Qualifiers
     source          1..8932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..47
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    164..185
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(200..205,206..223,224..230)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    238..254
                     /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     243..265
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     262..278
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     262..278
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4235..4246
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     regulatory      4451..4460
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4457..4459,4460..4462,4463..5176)
                     /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(4457..4459,4460..4462,4463..5173)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4502..4523)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4763..4782)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5110..5131
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(5201..5225)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5251..5292
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(6118..6135)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6118..6134)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6127..6149)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6347..6802
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6434..6453)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6644..6665
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6819..6838)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6938..6960
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6998..7016)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7084..7188
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7189..7257,7258..8049)
                     /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(7407..7426)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8220..8808
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8709..8728
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
       61 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
      121 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
      181 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
      241 tgagcggata acaatttcac acaggaaaca gctatgacat gattacgaat tcgaatggcc
      301 atgggacgtc gacgcttgca tgcctgcagc gtttccgata cccccttata tcagcacaca
      361 ttcagtcatc ataccacaaa atgttgtgat agtgataggc aaaactaccg tatttcctct
      421 attaatcttg catgcaagac taattttcga ttaacccgta ggggtgcaag actaatagag
      481 actgcaagac tattagaggc tgaaatacta attttcgtat gctcaataat tttggaaatt
      541 ggcctatttt ttgtagaaac ttgataccgt ttaaacaagg aaaaatacac actttttaat
      601 atttcatcaa taatttgaac gattttgtga ttttaagttc aatttgccca aaaaaagaca
      661 atttttctga cgttaccgca aagtaatgct ggtcaggcaa aaagagcggt gcaaaaatat
      721 aagagactgc aatactaata gaggaaatac ggtaattgaa ttttagtgaa acttgcgaca
      781 gttttcctca tttttttgtt attccggcat acgagtgtgg catacgagtg aggtcatctt
      841 ttgtttcttc cgtttcttca tcgcttttga aaaaaaatgt tgaagaaaac ttcgtagccg
      901 atagccggat aatgtttggc acggcgttcc aacatatatc attggaaatt ttttaagatt
      961 tttgcgaaaa atcacattct tcacgatgag aacacgttat tgaaggaata taaatcaaga
     1021 ataacatata gttatatttc tctattactt taacgttaaa tatgagcaaa tttgagcatt
     1081 ttgattgcga tgaaaagcag aatcgagtca actgaaatcc gttcaaaaac ctaagctcgg
     1141 ttcgcccgaa agtcgatttt tccaatagcc gaacagcact gcttctttat tttccaggta
     1201 tttttggggt gtaagaatta tatcttcttt gtttgggtat ttacgtttcc gaattccctt
     1261 tcttgatctg tcgctatttt tcagaaacca gaacagttta tcctttttca tgagaacctg
     1321 aatagctcaa aaacccgttt aatttctttt catcatctca tttgaaactt tcctagtctt
     1381 cttaatgatt tccaggctcc tcctacattt gtatctcaag atgcatacac tttatttccg
     1441 ctccaattcg aatcatctac ttctcttttt tcttcaactt tctacttttt tccattcttt
     1501 tctttcaaat gttcgacgtc ttcaagtgac tgctcttcat tctcctcatt tttctcgaat
     1561 ttcattcttg tctctttttt gctcaaaaat ggaaaatgaa agtgacgtga gatttggacg
     1621 gcgggacacg ggggcagtag aagcagcaaa aaggagagaa agaggacaca aataagaaga
     1681 acgaattcaa aaataagcgg agaggagcta tttccgtcaa ttctacctcc ccaatcttca
     1741 tcaattcggc tcaattgaat gacgtcacag ggagataaac cgtttggatg agcgccgtcc
     1801 atcactgacg ccatcccgtt tgggacaaga aaagagaaaa aagagcacaa agtttttggt
     1861 gacggatctt gtcaatcata tgaaagttgt tctgattgat tgtcagtttt tttcctactt
     1921 ttttggattc tatccacttc tgaacttttg acaagtttca aactttctga atcattttct
     1981 atgcattttc ctggaattct ttttatgtaa aatatgaaat agaatgtttt tgaattcaag
     2041 ttctgctttt ttcttctttt ctgttctgtt cgggcttggg tatgcttttt ttaaaaatta
     2101 ttttgcacat cgaccaataa gtgcgcaact tataaaatta atttattttt gttaattttt
     2161 gaaatacttg tattgcttta agtgatctga cctcgccctg agctttccac gtagttatca
     2221 aatacaaatc ctccaagggt aacgtaccta tattactgat ctttataata actttatcac
     2281 ctgtccagtt ccagaggaat tctgttaagc ttataggcac agaaggagtc atttctgctg
     2341 gtttttaatg atccaaatct ttatttcaag taaaaaactg aacacttgcg aataaaacta
     2401 tcagattaac cattcaccaa aaatgtgttt gaatctaaaa ctttctcagt attccaaata
     2461 tagaaataaa taaccacgac attgctaaaa tctgtctgaa ttgtgtactc cttaccgtga
     2521 agtaataatg gatataatga atcgtttgaa atgaatgatc agcacatttt tggtgaaaga
     2581 tcacaaataa ggaataagcg tcggaaaata aaacgattat ttcggatcaa aaatttgttg
     2641 aagatcatat acactcgaga ccaagattat tctagacaat tttcaaattg gcttctttgt
     2701 ttgcaaatct tcataataat ctgtgaagtt tggaaatttg aatttttaat cttttttcat
     2761 agattatagt ttttatttct ttgcaaaact atattaaaaa ccgatgcatt gttttaggga
     2821 aattaatgag ccttttgttc aacactaaaa acaataaaat taaaattttg gcttcatcat
     2881 ttgacctttt ttaagttcga aaacttttct cgtatttctg aaccgccaat tttttcacac
     2941 atctctagac ttttggtgcc cgttccagaa agttaagtaa ttgctattct aagaaagttc
     3001 tcaacattgt tttttagttc tgattgaatt ctgatgttcc aggaatatat tttaaattta
     3061 atattctttg cactatttct atactaacta aataataaat agtctaagta tgttcaatga
     3121 gaacaaaaaa gctctatcta tatttttcta tcctatttca tttatatcct tttcattttg
     3181 aactcaccag cttctcttct tcttcttgtc catatacttc ttaaaagtct cgaacttctc
     3241 tctctcactc tccatcaatt tctttatggc taccgcttgc gctctgccgc ttccgaagaa
     3301 agaaggcgtt ggcagagctc tcaattctat tttttctcgc cgtaggctat cgatttctca
     3361 gcttctcttc tctcttcctc actccaccac caccacttcg ggcttcttct tttcttcatt
     3421 ttcgtcttct tcttcatcct cttctttttt tttcagttcc ccctcactcc tccccttata
     3481 tgcgcgtgcg agaggggtgc aaaagcagcg cgcatccgag aattgaaacg agaaaacgga
     3541 gacgcagcag cagttcgtcc tcagaaaaat agccagtaaa aagagaaaaa gatagagaga
     3601 gacctatcgc attttatttt caaattgcaa ttcctgcaat tcatgtgtgc gtgtgcccat
     3661 tttcaattct tcccgttatt tttcagacca ttaccaacgt tctttctaga tcttgagaca
     3721 atcttccttc tgctcaatcg ttcgtcgtga agacggcatc gacgacgacg acgatcagca
     3781 gtaaaggatc cttatgaacg ctataatctt ctgtctcctc ttcacaactg tcactgccac
     3841 ttatgaagtt ttcggaaaag gaatagaaca cagaaatgaa catttgatca tcaatcaact
     3901 tgatatcata ccagttgagt caactccaac tccaaaccgt gcctcaagag tccagaaacg
     3961 tctatgcgga agacgtctta ttttattcat gcttgcaaca tgtggagaat gtgatacagg
     4021 tacgactttt tttttaagta ttaaaattat ttttgaacgt aaaactttaa tggaaattca
     4081 ttcaattcta gaaaattttg agttaataaa atgattacta ttaaaaatta aattttgaaa
     4141 ttgtcaattt attctggaaa gattttttgt ttctgaaaat taatacttca acaaaacgcg
     4201 ccgtacatgt ctacgcattg catgcgttga tcgcattgaa ggcaggcata catttataag
     4261 aattttcagg acgtcaaaaa gtccaactat tgtttttcat actgtttcaa acgttcaccc
     4321 tttaaacata tgatacattt cagattcatc agaagacctt tcgcatattt gctgcataaa
     4381 acaatgtgac gttcaagata tcatcagagt ctgctgcccg aattcattta gaaaacggga
     4441 tccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     4501 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     4561 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     4621 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     4681 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     4741 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     4801 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     4861 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     4921 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     4981 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     5041 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     5101 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     5161 cgagctgtac aagtaaagcg gccgcactcg agatatctag acccagcttt cttgtacaaa
     5221 gtggttgatc tagagggccc gcggttcgaa ggtaagccta tccctaaccc tctcctcggt
     5281 ctcgattcta cgcgtaccgg ttagtaatga gtttatctga gctccgcatc ggccgctgtc
     5341 atcagatcgc catctcgcgc ccgtgcctct gacttctaag tccaattact cttcaacatc
     5401 cctacatgct ctttctccct gtgctcccac cccctatttt tgttattatc aaaaaacttc
     5461 tcttaatttc tttgtttttt agcttctttt aagtcacctc taacaatgaa attgtgtaga
     5521 ttcaaaaata gaattaattc gtaataaaaa gtcgaaaaaa attgtgctcc ctccccccat
     5581 taataataat tctatcccaa aatctacaca atgttctgtg tacacttctt atgtttttta
     5641 cttctgataa atttttttga aacatcatag aaaaaaccgc acacaaaata ccttatcata
     5701 tgttacgttt cagtttatga ccgcaatttt tatttcttcg cacgtctggg cctctcatga
     5761 cgtcaaatca tgctcatcgt gaaaaagttt tggagtattt ttggaatttt tcaatcaagt
     5821 gaaagtttat gaaattaatt ttcctgcttt tgctttttgg ggtttcccct attgtttgtc
     5881 aagatttcga ggacggcgtt tttcttgcta aaatcacaag tattgatgag cacgatgcaa
     5941 gaaagatcgg aagaaggttt gggtttgagg ctcagtggaa ggtgagtaga agttgataat
     6001 ttgaaagtgg agtagtgtct atggggtttt tgccttaaat gacagaatac attcccaata
     6061 taccaaacat aactgtttct actagtggcc acgtgggccg tgcaccttaa gcttggcact
     6121 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     6181 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     6241 ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac
     6301 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     6361 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     6421 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     6481 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     6541 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     6601 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     6661 actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg
     6721 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     6781 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     6841 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     6901 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     6961 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     7021 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     7081 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     7141 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     7201 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     7261 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     7321 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     7381 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     7441 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     7501 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     7561 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     7621 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     7681 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     7741 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     7801 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     7861 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     7921 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     7981 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     8041 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     8101 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     8161 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     8221 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     8281 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     8341 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     8401 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     8461 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     8521 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     8581 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     8641 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     8701 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     8761 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     8821 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     8881 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga ta
//
