LOCUS       sequence-388329-        6177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of PACLight1 biosensor for PACAP (Pituitary 
            Adenylate Cyclase Activating Peptide).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6177)
  AUTHORS   Cola RB, Niethammer SN, Rajamannar P, Gresch A, Bhat MA, Assoumou K,
            Williams ET, Hauck P, Hartrampf N, Benke D, Stoeber M, Levkowitz G, 
            Melzer S, Patriarchi T
  TITLE     Probing PAC1 receptor activation across species with an engineered 
            sensor.
  JOURNAL   Elife. 2024 Aug 15;13:RP96496. doi: 10.7554/eLife.96496.
  PUBMED    39145773
REFERENCE   2  (bases 1 to 6177)
  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: "2024-08-15"; volume: "13:RP96496. doi"; pages: " 
            10.7554/eLife.96496"
COMMENT     Sequence Label: pCMV_PACLight1
FEATURES             Location/Qualifiers
     source          1..6177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..206
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     157..177
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1563..1584
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1690..1711)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1951..1970)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2603..2724
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2640..2659)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2694..2713
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2731..3186)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2868..2889)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3080..3099
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3213..3317
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3319..3676
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3319..3510
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3342..3362)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3527..3662
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3711..4505
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3711..4505
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3765..3784)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4375..4394
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4693..4712)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4737..4784
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5113..5701
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5602..5621
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        join(5876..6177,1..2)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
       61 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      121 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      181 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc
      241 ggactcagat ctcgagctca agcttggatc cggtacctgc gcgccatgaa gacgatcatc
      301 gccctgagct acatcttctg cctggtgttc gccatggcgg gcgtagtgca cgtttccctt
      361 gctgcactgc tcctcttgcc aatggcgcca gccatgcaca gtgactgcat ctttaagaag
      421 gaacaagcta tgtgtctcga aaaaatccaa agagctaatg agcttatggg ctttaatgat
      481 tcttcaccag gttgcccggg tatgtgggat aacattacat gttggaagcc cgcgcacgta
      541 ggtgaaatgg tattggtttc ttgcgctgaa ctgttccgaa tttttaaccc cgatcaagta
      601 tgggaaacgg aaacgattgg tgaaagtgac ttcggagact caaatagtct ggatctctca
      661 gatatgggtg tggtctctcg gaattgtacc gaggatggct ggtccgagcc gtttccgcat
      721 tattttgacg catgtggttt cgacgaatac gaatctgaga ccggagatca agattactac
      781 taccttagtg taaaggcact gtatacagtt ggttacagta caagtctggt tacactcact
      841 acagctatgg taatcttgtg cagattcagg aagctccatt gtacgcgaaa tttcattcac
      901 atgaatttgt tcgtgtcttt tatgctccgg gctatttccg ttttcattaa agactggatt
      961 ctgtacgcag agcaggattc aaaccattgt ttcatcagta cagtagaatg taaggcagta
     1021 atggtttttt ttcattactg cgtggtttcc aactactttt ggctgtttat agaaggtttg
     1081 tatctgttca cacttctggt tgaaactaag aagaaggagc gacggtactt ctactggtac
     1141 acaattatag gatggggcac tccgaccgtc tgtgtgacag tatgggcgac actccggctt
     1201 tacttcgacg acacaggttg ttgggacatg aacgattcca cggctctctg gtgggtaata
     1261 aaggggccag tcgtggggag cattatggtt aattttgtcc tctttatcgg cattatagta
     1321 atccttgtcc agaagctgca gctgagctca ctcattaacg tctatatcaa ggccgacaag
     1381 cagaagaacg gcatcaaggc gaacttcaag atccgccaca acatcgagga cggcggcgtg
     1441 cagctcgcct accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1501 gacaaccact acctgagcgt gcagtccaaa ctttcgaaag accccaacga gaagcgcgat
     1561 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1621 tacaagggcg gtaccggagg gagcatggtg agcaagggcg aggagctgtt caccggggtg
     1681 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1741 gagggtgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1801 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1861 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1921 tacatccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1981 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2041 gaggacggca acatcctggg gcacaagctg gagtacaaca atcatgacca actgaatgag
     2101 agttctatat acttgcgcct cgcgcgaagt acactcctgc tgatccctct ttttggtata
     2161 cattacacag tcttcgcgtt tagtccagag aacgtcagta aacgcgaaag actcgtcttc
     2221 gaactggggt tgggttcatt tcaggggttc gtcgttgcag tactttattg ctttctgaat
     2281 ggcgaagtcc aagcagaaat taagcggaag tggcgctctt ggaaggtcaa tcgctatttc
     2341 gccgtggact tcaaacatcg ccatcccagc ttggcttcct caggcgtgaa tgggggcacg
     2401 cagttgagta ttctgtcaaa gagttcatcc cagattagaa tgtcaggtct cccggccgac
     2461 aacttggccg catgatgtac agcggccgcg actctagatc ataatcagcc ataccacatt
     2521 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     2581 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     2641 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     2701 gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat
     2761 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     2821 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     2881 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     2941 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     3001 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     3061 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     3121 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     3181 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3241 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3301 attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg
     3361 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3421 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3481 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3541 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3601 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3661 cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac
     3721 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3781 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3841 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg
     3901 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3961 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4021 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4081 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4141 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4201 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc
     4261 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4321 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4381 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4441 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4501 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     4561 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4621 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag
     4681 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
     4741 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg
     4801 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg
     4861 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc
     4921 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga
     4981 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     5041 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5101 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5161 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5221 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5281 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5341 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5401 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5461 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5521 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5581 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5641 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5701 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5761 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt
     5821 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     5881 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     5941 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     6001 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     6061 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     6121 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attacca
//
