LOCUS       sequence-454989-        5928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Modular BxB1 landing pad vector containing C-term mNeonGreen11 tag, 
            puroR marker, and a cAMP GFP reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5928)
  AUTHORS   Howard MK, Hoppe N, Huang XP, Mitrovic D, Billesbolle CB, Macdonald 
            CB, Mehrotra E, Rockefeller Grimes P, Trinidad DD, Delemotte L, 
            English JG, Coyote-Maestas W, Manglik A
  TITLE     Molecular basis of proton sensing by G protein-coupled receptors.
  JOURNAL   Cell. 2025 Feb 6;188(3):671-687.e20. doi: 
            10.1016/j.cell.2024.11.036. Epub 2025 Jan 2.
  PUBMED    39753132
REFERENCE   2  (bases 1 to 5928)
  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: 
            "10.1016/j.cell.2024.11.036"; journalName: "Cell"; date: "2025-02-6-
            6"; volume: "188"; issue: "3"; pages: "671-687.e20"
COMMENT     Sequence Label: pLP_mod_mNeonGreen11_P2A_puroR_CRE-GFP
FEATURES             Location/Qualifiers
     source          1..5928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..28)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12..28)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(25..47)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    36..52
                     /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)."
     promoter        complement(join(60..66,67..84,85..90))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    105..126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(243..260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(414..1002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(494..513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1173..1964,1965..2033))
                     /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     1796..1815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2034..2138)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2164..2619)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2301..2322)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2513..2532
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2746..2768
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2760..2777
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2761..2777
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2784..2803
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2784..2802
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2876..2913
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             3048..3104
                     /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"
     polyA_signal    3700..3924
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4329..4360
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             4396..4416
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4432..4434,4435..4437,4438..5148)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4432..4434,4435..4437,4438..5148)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4477..4498)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4738..4757)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5085..5106
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5746..5867
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5783..5802)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5837..5856
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(5876..5894)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
       61 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
      121 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
      181 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
      241 tcctcgctca ctgactcgct gcactcggtc gttcggctgc ggcgagcggt atcagctcac
      301 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      361 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      421 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      481 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      541 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      601 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      661 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      721 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      781 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      841 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      901 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
      961 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1021 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1081 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     1141 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1201 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1261 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1321 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1381 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1441 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1501 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1561 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1621 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1681 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1741 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1801 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1861 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1921 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1981 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2041 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     2101 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     2161 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     2221 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2281 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2341 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2401 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2461 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2521 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2581 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     2641 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     2701 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     2761 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccgggc
     2821 cccccctcga ggtcgacgat gtaggtcacg gcattccgga ctcagcgctc gagccggctt
     2881 gtcgacgacg gcggtctccg tcgtcaggat catccgcaga ttataccgca actacacgcc
     2941 accccgtcag atccggccac catcgagacg ggaacgtctc agggtcggga tctggttcaa
     3001 ccgagctcaa cttcaaggag tggcaaaagg cctttaccga tatgatggca actaatttta
     3061 gtctactgaa acaagctggt gatgtggagg aaaatccagg accaaccgag tacaagccca
     3121 cggtgcgctt agccacccgc gacgacgtcc ccagggccgt acgcaccttg gcagctgctt
     3181 tcgccgacta ccccgctacg agacacaccg ttgatcctga ccgccacatc gaaagagtta
     3241 ccgagttgca agaattgttc ttgacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     3301 cagacgacgg cgccgctgtg gcggtatgga ccacgccgga gagcgtagaa gctggagcgg
     3361 tattcgctga gatcggaccg cgcatggcag aattgagcgg ttcccggctg gccgcacagc
     3421 aacagatgga aggactcctg gctcctcacc ggcccaagga gcctgcatgg ttcctggcaa
     3481 ccgttggtgt gtcgcctgac caccagggaa agggtctggg cagcgctgtt gtgctccctg
     3541 gagtggaggc ggcagaaaga gctggagtgc cagcattcct ggaaacctcc gctccccgca
     3601 acctcccctt ctacgaaaga ctcggcttca ccgttaccgc agacgtcgag gttcccgaag
     3661 gaccgagaac ctggtgcatg acccgcaagt aaggcccgac tgtgccttct agttgccagc
     3721 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3781 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3841 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3901 ctggggatgc ggtgggctct atggcacttt tatactttta tgagggacag ccccccccca
     3961 aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg
     4021 ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc
     4081 ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg
     4141 agcctgcaga cacctggggg gatacgggga aaaggacgcg tacgcgtgca ccagacagtg
     4201 acgtcagctg ccagatccca tggccgtcat actgtgacgt ctttcagaca ccccattgac
     4261 gtcaatggga gaactcccta cggtaccgag gatatcaaga tctggcctcg gcggccaagc
     4321 ttagacacta gagggtatat aatggaagct cgacttccag cttggcaatc cggtactgtt
     4381 ggtaaagcca ccatgcccaa gaagaagagg aaggtggagg acgccggttc tatggtgagc
     4441 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     4501 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     4561 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     4621 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     4681 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     4741 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     4801 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     4861 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     4921 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     4981 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     5041 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     5101 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg atccatatca
     5161 ctgatcgctg cactcgcggt tgattatgta ataggcatgg aaaacgcgat gccctggaac
     5221 ctgccagcag acctcgcatg gtttaagcgg aacactctga ataagccagt cattatgggc
     5281 cgacatacgt gggaaagcat tggacgccca ctgccaggaa ggaagaatat aatcctgtca
     5341 tcacagccgt ccacggacga tagagtcact tgggtaaaat ccgttgacga agccatagct
     5401 gcctgtggtg atgtgcctga gataatggta ataggaggag ggcgggtaat cgaacaattt
     5461 ctgcctaaag cccagaagct gtaccttacc catattgacg cggaagtaga gggcgacaca
     5521 catttcccag attatgagcc agatgattgg gagagcgttt tctccgaatt tcatgatgca
     5581 gacgcccaga atagccacag ctactgcttt gaaatattgg agcgccgata agggatcctg
     5641 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     5701 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     5761 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5821 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttagga gatccccttt
     5881 agtgagggtt aatttaagct tgcggccgca ctcgagaatt cgagcttg
//
