LOCUS       sequence-334863-        9069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a mu-opioid receptor-based circular permuted green 
            fluorescent protein sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9069)
  AUTHORS   Kroning KE, Wang W
  TITLE     Designing a Single Protein-Chain Reporter for Opioid Detection at 
            Cellular Resolution.
  JOURNAL   Angew Chem Int Ed Engl. 2021 Mar 4. doi: 10.1002/anie.202101262.
  PUBMED    33662184
REFERENCE   2  (bases 1 to 9069)
  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: "Angew Chem 
            Int Ed Engl. 2021 Mar 4. doi: 10.1002/anie.202101262."
COMMENT     Sequence Label: M-SPOTIT1.1
FEATURES             Location/Qualifiers
     source          1..9069
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        46..272
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             273..453
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    500..625
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1118..1351
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1536..1580
                     /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    1888..2005
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2122..2425
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2426..2629
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2580..2600
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4045..4066
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4172..4193)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4433..4452)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    4967..5555
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5020..5040)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5438..5449)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5627..5860
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5938..6059
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5975..5994)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6029..6048
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6099..6234
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6254..6273)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6255..6273)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6283..6300)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6283..6299)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6292..6314)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6441..6896
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6528..6547)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6738..6759
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6922..7026
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7027..7095,7096..7887)
                     /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(7245..7264)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8058..8646
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8547..8566
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8800..8817
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8934..8955
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8970..8975,8976..8993,8994..9000)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9008..9024
                     /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     9013..9035
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9032..9048
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9032..9048
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     join(9067..9069,1..18)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        join(9069,1..18)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa
       61 tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga
      121 aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa
      181 ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata
      241 ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag
      301 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
      361 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
      421 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
      481 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
      541 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
      601 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
      661 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
      721 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
      781 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
      841 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
      901 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
      961 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     1021 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     1081 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     1141 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     1201 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     1261 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     1321 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     1381 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     1441 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     1501 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     1561 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     1621 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     1681 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     1741 ccccgagagc gctgccgtct ggaccgatgg ctgtgtggaa gtgctcgccg atagtggaaa
     1801 cagacgcccc agcactcgtc ctagggcaaa ggatctgcag taatgagaat tctagatctt
     1861 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     1921 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     1981 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg
     2041 gcgccggctc gagggggccc ggggttatta atagtaatca attacggggt cattagttca
     2101 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     2161 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     2221 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     2281 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     2341 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     2401 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     2461 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     2521 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     2581 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac
     2641 tgtcgggatc caccgctagc ccaccatgag accccagatc ctgctgctcc tggccctgct
     2701 gaccctaggc ctggctgact acaaggacga tgatgacgcc atggactctt ccgctgctcc
     2761 caccaatgcc tcaaattgta cagacgcctt ggcctatagc tcttgctctc ccgcaccatc
     2821 tccgggaagc tgggttaacc tcagtcatct cgacgggaac ctgtccgacc cttgtggccc
     2881 taataggacc gacctcggtg gtcgagatag cctgtgcccc cctactggaa gccccagcat
     2941 gatcacagct atcacaataa tggctctcta tagtatcgtc tgcgtcgtgg gactctttgg
     3001 caattttctc gtcatgtacg ttatcgtcag atacacaaag atgaaaaccg caaccaatat
     3061 ttacatcttt aatctggccc tggcggatgc ccttgcaacc agtacactgc cgtttcaatc
     3121 cgtgaattat ctgatgggca catggccctt cggaaccact ctgtgcaaaa tcgtaatctc
     3181 aattgattat tataacatgt tcacctccat ttttaccttg tgtacaatgt cagtagaccg
     3241 ctatatcgcc gtatgccacc ctgtcaaagc tcttgatttc aggaccccta ggaatgccaa
     3301 gatcatcaat gtgtgcaatt ggattctgtc tagcgctatc ggcttgccgg tgatgttcat
     3361 ggccactacc aagtacagac aaggttcaat cgactgtacc ctcacatttt cacatcccac
     3421 gtggtattgg gagaacctgc tgaagatctg tgtgttcatc ttcgccttta tcatgccagt
     3481 actgataatc accgtttgtt atggtcttat gatcctgcgc ctgaagagcg tgcgcatgct
     3541 cagcggctcc aaagaaaagg accggaacct tcggcgaatc acacggatgg tcctggtggt
     3601 tgtggctgtg ttcattgttt gctggacccc tattcatatt tatgttataa ttaaggccct
     3661 ggtgaccatc ccggaaacca cattccagac cgtttcctgg cacttttgta tagcattggg
     3721 atataccaac agttgcctta atcccgttct gtacgcgttt ctggacgaga atttcaagcg
     3781 atgcttcagg gagttctgct tccccctcaa gatgcgcatg gagcggcagg gcgcgcccaa
     3841 cgtctatatc aaggccgaca agcagaagaa cggcatcaag gcgaacttcc acatccgcca
     3901 caacatcgag gacggcggcg tgcagctcgc ctaccactac cagcagaaca cccccatcgg
     3961 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc gtgcagtcca aactttcgaa
     4021 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     4081 cactctcggc atggacgagc tgtacaaggg cggtaccgga gggagcatgg tgagcaaggg
     4141 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     4201 ccacaagttc agcgtgtccg gcgagggtga gggcgatgcc acctacggca agctgaccct
     4261 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     4321 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     4381 caagtccgcc atgcccgaag gctacatcca ggagcgcacc atcttcttca aggacgacgg
     4441 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     4501 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     4561 ccttaaggaa gatatcatgg ctcaagtcca acttgtagag tctggaggag gtctcgtgcg
     4621 gcccggcggt agcctgcgac tctcctgtgt tgattcagag aggacgagct accctatggg
     4681 gtggtttaga cgggcacccg gaaaggagcg cgaattcgtt gcaagtatca cgtggagtgg
     4741 cattgacccg acttatgctg atagcgttgc cgaccggttt actacgtcca gagacgttgc
     4801 gaataacacg ctgtacctcc agatgaactc tctcaagcac gaagatacag cggtctatta
     4861 ctgtgctgca agagcacccg tcggtcaatc ttcctctcct tatgattatg actattgggg
     4921 acaggggact caggtaactg tgtccagtta gacgcgttaa gtcgacaatc aacctctgga
     4981 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     5041 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     5101 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     5161 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     5221 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     5281 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     5341 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     5401 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     5461 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     5521 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
     5581 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     5641 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     5701 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     5761 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     5821 tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag tagttcatgt
     5881 catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac
     5941 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     6001 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     6061 catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca
     6121 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     6181 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga
     6241 cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     6301 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     6361 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     6421 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     6481 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     6541 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     6601 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     6661 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     6721 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     6781 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     6841 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     6901 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6961 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     7021 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     7081 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     7141 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     7201 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     7261 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     7321 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     7381 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     7441 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     7501 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     7561 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     7621 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     7681 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     7741 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     7801 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     7861 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     7921 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     7981 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     8041 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     8101 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     8161 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     8221 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     8281 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     8341 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     8401 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     8461 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     8521 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     8581 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     8641 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     8701 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     8761 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     8821 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     8881 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     8941 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     9001 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     9061 aagcgcgca
//
