LOCUS       sequence-501616-        6286 bp    DNA     circular SYN 03-MAR-2026
DEFINITION  Expresses full length corticotropin releasing factor peptide caged 
            by chemically activated protein domains in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6286)
  TITLE     A chemogenetic approach for temporal and cell-specific activation of
            endogenous GPCRs in vivo
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6286)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 3, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: CRF-CapC
FEATURES             Location/Qualifiers
     source          1..6286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        312..515
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     466..486
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     560..579
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        560..578
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             829..1149
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1156..1182
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1183..1209
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1957..2466
                     /codon_start=1
                     /product="N-terminal portion of the CIB1 transcription 
                     factor from Arabidopsis thaliana"
                     /label=CIBN
                     /note="binds to CRY2 (cryptochrome 2) that has absorbed 
                     blue light (Kennedy et al., 2010)"
                     /translation="MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSS
                     MITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKAAKFDTETKDCN
                     EAAKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKE
                     LEKTDYI"
     CDS             2479..2535
                     /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"
     regulatory      2560..2569
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2566..3276
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2711..2729)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2979..2998
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3307..3324)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3313..3520
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3549..3689
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3549..3678
                     /label=AAV2 ITR
     rep_origin      3764..4219
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3851..3870)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4061..4082
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4236..4255)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4355..4377
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4415..4433)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4501..4605
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4606..4674,4675..5466)
                     /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(4824..4843)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5637..6225
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6126..6145
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttgtaca tagtaatcaa ttacggggtc attagttcat
      181 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggcattatg
      241 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      301 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      361 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      421 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      481 ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
      541 cttactggct tatcgaaatt aatacgactc actataggga gacccaagct tgccaccatg
      601 aagaccatca tcgccctgag ctacatcttc tgcctggtgt tcgccagcga agaaccgccg
      661 attagcctgg atctgacctt tcatctgctg cgcgaagtgc tggaaatggc gcgcgcggaa
      721 cagctggcgc agcaggcgca tagcaaccgc aaactgatgg aaattattcc taatttgttt
      781 gggtatgtgt caggcggttc tggtactgga tctggttctg gaggttctgg agtgcaggtg
      841 gaaaccatct ccccaggaga cgggcgcacc ttccccaagc gcggccagac ctgcgtggtg
      901 cactacaccg ggatgcttga agatggaaag aaagttgatt cctcccggga cagaaacaag
      961 ccctttaagt ttatgctagg caagcaggag gtgatccgag gctgggaaga aggggttgcc
     1021 cagatgagtg tgggtcagag agccaaactg actatatctc cagattatgc ctatggtgcc
     1081 actgggcacc caggcatcat cccaccacat gccactctcg tcttcgatgt ggagcttcta
     1141 aaactggaaa gatcttaccc atacgatgtg ccagattacg cctatcccta tgacgtccct
     1201 gattacgctc tcgacttcca gaaggcctcc agcatagtct ataagaaaga gggggaacag
     1261 gtggagttct ccttcccact cgcctttaca gttgaaaagc tgacgggcag tggcgagctg
     1321 tggtggcagg cggagagggc ttcctcctcc aagtcttgga tcacctttga cctgaagaac
     1381 aaggaagtgt ctgtaaaacg ggttacccag gaccctaagc tccagatggg caagaagctc
     1441 ccgctccacc tcaccctgcc ccaggccttg cctcagtatg ctggctctgg aaacctcacc
     1501 ctggcccttg aagcgaaaac aggaaagttg catcaggaag tgaacctggt ggtgatgaga
     1561 gccactcagc tccagaaaaa tttgacctgt gaggtgtggg gacccacctc ccctaagctg
     1621 atgctgagct tgaaactgga gaacaaggag gcaaaggtct cgaagcggga gaaggcggtg
     1681 tgggtgctga accctgaggc ggggatgtgg cagtgtctgc tgagtgactc gggacaggtc
     1741 ctgctggaat ccaacatcaa ggttctgccc acatggtcca ccccggtgca gccaatggcc
     1801 ctgattgtgc tggggggcgt cgccggcctc ctgcttttca ttgggctagg catcttcttc
     1861 tgtgtcaggt gccggcaccg aaggcgcaag ggctcgggct cgacctcggg ctcgggcagc
     1921 ggtggctcta gaggttctgg tggcagctct ggaggtatga atggagctat aggaggtgac
     1981 cttttgctca attttcctga catgtcggtc ctagagcgcc aaagggctca cctcaagtac
     2041 ctcaatccca cctttgattc tcctctcgcc ggcttctttg ccgattcttc aatgattacc
     2101 ggcggcgaga tggacagcta tctttcgact gccggtttga atcttccgat gatgtacggt
     2161 gagacgacgg tggaaggtga ttcaagactc tcaatttcgc cggaaacgac gcttgggact
     2221 ggaaatttca aggcagcgaa gtttgataca gagactaagg attgtaatga ggcggcgaag
     2281 aagatgacga tgaacagaga tgacctagta gaagaaggag aagaagagaa gtcgaaaata
     2341 acagagcaaa acaatgggag cacaaaaagc atcaagaaga tgaaacacaa agccaagaaa
     2401 gaagagaaca atttctctaa tgattcatct aaagtgacga aggaattgga gaaaacggat
     2461 tatattcatg gaagcggagc tactaacttc agcctgctga agcaggctgg agacgtggag
     2521 gagaaccctg gacctaccgg tgctcgggat cctccggtcg ccaccatggt gagcaagggc
     2581 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     2641 tccgtgaacg gccacgagtt cgagattgaa ggtgagggag agggccgccc ctacgagggc
     2701 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     2761 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     2821 gactacttga agctgtcctt ccccgaggga ttcaagtggg agcgcgtgat gaacttcgag
     2881 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     2941 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     3001 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     3061 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     3121 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     3181 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     3241 cactccaccg gcggcatgga cgagctgtac aagtaagaat tcctcgagct agagctcgct
     3301 gaccagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     3361 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3421 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3481 agggggagga ttgggaagac aatagcaggc atgctgggga ggtaaccacg tgcggaccga
     3541 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     3601 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     3661 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     3721 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     3781 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3841 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3901 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3961 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4021 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4081 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     4141 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4201 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4261 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4321 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4381 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     4441 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     4501 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4561 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4621 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4681 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4741 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4801 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     4861 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4921 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4981 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5041 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5101 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5161 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5221 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5281 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     5341 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5401 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5461 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     5521 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5581 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5641 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5701 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5761 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5821 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5881 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5941 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6001 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6061 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6121 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6181 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6241 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
