LOCUS       sequence-420400-        7715 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Loss of function variant of uMASS_1 opioid sensor. AAV virus 
            production for Cre dependent expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7715)
  AUTHORS   Rappleye M, Wait SJ, Lee JD, Siebart JC, Torp L, Smith N, Muster J, 
            Matreyek KA, Fowler DM, Berndt A
  TITLE     Optogenetic Microwell Array Screening System: A High-Throughput 
            Engineering Platform for Genetically Encoded Fluorescent Indicators.
  JOURNAL   ACS Sens. 2023 Nov 24;8(11):4233-4244. doi: 
            10.1021/acssensors.3c01573. Epub 2023 Nov 13.
  PUBMED    37956352
REFERENCE   2  (bases 1 to 7715)
  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: 
            "10.1021/acssensors.3c01573"; journalName: "ACS Sens"; date: 
            "2023-11-24- 24"; volume: "8"; issue: "11"; pages: "4233-4244"
COMMENT     Sequence Label: pAAV_EF1a_DIO_HA/FLAG_muMASS_LF
FEATURES             Location/Qualifiers
     source          1..7715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     522..542
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(599..632)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(683..716)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1353..1372
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1612..1633
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1739..1760)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2855..2888
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2939..2972
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2997..3585
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3050..3070)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3468..3479)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3617..4093
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3816..3835)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4133..4273
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4133..4262
                     /label=AAV2 ITR
     rep_origin      4348..4803
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4435..4454)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4645..4666
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4820..4839)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4939..4961
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4999..5017)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5085..5189
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5190..5258,5259..6050)
                     /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(5408..5427)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6221..6809
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6710..6729
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6871..7011
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6882..7011
                     /label=AAV2 ITR
     promoter        join(7112..7715,1..574)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          join(7343..7715,1..565)
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
ORIGIN
        1 cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg
       61 gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg tgcctggcct
      121 cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc
      181 gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat ggaggacgcg
      241 gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc
      301 agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt
      361 ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt
      421 tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc
      481 cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt
      541 tcaaagtttt tttcttccat ttcaggtgtc gtgaggtacc ggatccgtcg actccggaat
      601 aacttcgtat aggatacttt atacgaagtt atgcagaatg gtagctggat tgtagctgct
      661 attagcaata tgaaacctct taataacttc gtatagcata cattatacga agttatggcg
      721 cgccttagcc gacttcgttc tcgtagcaga aagcggcggc tggtgctccc acgttgatgt
      781 cgatctggtc cagagggatg tactcgccct cgcttgtgat tctgctctta cctccgccgg
      841 gcaatggagc agtttctgcc tccagatttt ctagctggtg gttagttcga tccactgtat
      901 tagccgtgga gggatgttcc ctagtgttct gacggactcg agtggagttt tgctgttcga
      961 tcgtggacga ggttgggatg cagaactctc tgaagcatcg cttgaagttt tcatccagga
     1021 aggcgtaaag aactggattc aggcagctgt tcgtgtaacc caaagcaatg cagaagtgcc
     1081 aggaaacggt ctgaaatgtg gtttctggaa tcgtgatcag cgctttgatg atgacgtaga
     1141 tgtggatggg ggtccagcag acgataaata cagccacgac caccagcacc atccgggtga
     1201 tcctgcgcag attcctgtcc ttttctttca gttggtcaac catgttgtac tccagcttgt
     1261 gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca
     1321 gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga
     1381 agatggtgcg ctcctggatg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct
     1441 tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg
     1501 tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt
     1561 aggtggcatc gccctcaccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt
     1621 ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatgc
     1681 tccctccggt accgcccttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     1741 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttcgaa agtttggact
     1801 gcacgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     1861 gctggtagtg gtaggcgagc tgcacgccgc cgtcctcgat gttgtggcgg atcttgaagt
     1921 tcgccttgat gccgttcttc tgcttgtcgg ccttgatata gacgttatca agtgagctca
     1981 ggcgaacgct cttgagtcgt aagatcatca ggccgtaaca cacagtgatg atgaggatcg
     2041 gcatgatgaa agcgaagata aagacacaga ttttgagcag gttctcccag taccaggttg
     2101 ggtgggagaa cgtgagggtg caatctatgg acccctgcct gtatttcgtg gttgccatga
     2161 acattacagg cagaccgatg gcagaagaga ggatccagtt gcagacgttg acgattttgg
     2221 catttcgggg ggtacggaaa tccagggctt tgactgggtg gcagacagca atgtagcggt
     2281 ccacgctcat ggtgcagagg gtgaatatgc tggtgaacat gttgtagtat cctattgaga
     2341 tcacgatctt gcagaggatg gttccgaagg gccatgttcc catcaggtag ttgacactct
     2401 gaaagggcag tgtactggtc gctaaggcgt ctgccagagc aaggttgaaa atgtagatgt
     2461 tggtggcagt cttcattttg gtgtatctta caatcacata catgaccagg aagtttccga
     2521 agaggcccac tacacacacg atagagtaga gggccatgat ggtaatggct gtgaccatgg
     2581 aagggctgcc ggtctgaggg cacaggctgt cgttcccgcc aagcccggtg cggttcagac
     2641 cgcatggatc ggactggttg ccatcaacgt gggacaagtt gagccaggag ccaggtgctg
     2701 gggagcaact tgcctgagct aaggggtctg agcagtcgct ggtgttccct gggccggtgc
     2761 tgctgtccat ggcgtcatca tcgtccttgt agtcggcgaa caccaggcag aagatgtagc
     2821 tcagggcgat gatcgtcttc atggtggcgc tagcataact tcgtataaag tatcctatac
     2881 gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaat
     2941 aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt atcgataatc
     3001 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     3061 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3121 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3181 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3241 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3301 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3361 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3421 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3481 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3541 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
     3601 tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
     3661 ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
     3721 gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg
     3781 gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc
     3841 agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc
     3901 tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
     3961 tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca
     4021 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
     4081 cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc
     4141 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4201 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4261 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4321 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4381 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4441 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4501 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4561 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4621 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     4681 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4741 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     4801 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     4861 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4921 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4981 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5041 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5101 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5161 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5221 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5281 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5341 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5401 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5461 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5521 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5581 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5641 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5701 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     5761 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     5821 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     5881 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     5941 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6001 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6061 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6121 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6181 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6241 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6301 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6361 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6421 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6481 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6541 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6601 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6661 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6721 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6781 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6841 tggccttttg ctggcctttt gctcacatgt cctgcaggca gctgcgcgct cgctcgctca
     6901 ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga
     6961 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccgca
     7021 cgcgtaagct ttgcaaagat ggataaagtt ttaaacagag aggaatcttt gcagctaatg
     7081 gaccttctag gtcttgaaag gagttctaga tggctccggt gcccgtcagt gggcagagcg
     7141 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta
     7201 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     7261 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     7321 cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt
     7381 tacgggttat ggcccttgcg tgccttgaat tacttccact ggctgcagta cgtgattctt
     7441 gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc
     7501 ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc
     7561 tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt
     7621 tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat
     7681 ctgcacactg gtatttcggt ttttggggcc gcggg
//
