LOCUS       sequence-495664-        6299 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  Plasmid used to express LplA's site-specific substrate LAP2 
            (13-amino acid acceptor peptide) in mammalian cells. CAAX targets it
            to the inner leaflet of the plasma membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6299)
  TITLE     Ting Lab LplA Plasmids 2025
REFERENCE   2  (bases 1 to 6299)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: His-LAP-BFP-CAAX
FEATURES             Location/Qualifiers
     source          1..6299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(1006..1008,1009..1011,1012..1722)
                     /codon_start=1
                     /product="blue variant of GFP"
                     /label=BFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1051..1072)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1312..1331)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1659..1680
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1852..1870)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1853..1870)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1890..1907)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1896..2120
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2166..2594
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2253..2272)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2463..2484
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2603..2623)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2608..2937
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2788..2923
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2850..2869
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3004..3798
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3058..3077)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3668..3687
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3972..4093
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4009..4028)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4063..4082
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4142..4158)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4142..4158)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4155..4177)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4166..4182
                     /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(4190..4196,4197..4214,4215..4220))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4235..4256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4373..4390)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4544..5132)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4624..4643)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5303..6094,6095..6163))
                     /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     5926..5945
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6164..6268)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag cggcttcgag
      961 atcgacaagg tgtggtacga cctggacgcc aaggatccga gctcgatggt gagcaagggc
     1021 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1081 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1141 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1201 agccacggcg tccagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1261 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1321 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1381 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1441 ttcaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1501 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1561 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca gccactacct gagcacccag
     1621 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1681 accgccgccg ggatcactct cggcatggac gagctgtaca agcgcagcaa gctgaacccc
     1741 cccgacgaga gcggccccgg ctgcatgagc tgcaagtgcg tgctgagcta agaattctgc
     1801 agatatccat cacactggcg gccgctcgag catgcatcta gagggcccta ttctatagtg
     1861 tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1921 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1981 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2041 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2101 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2161 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2221 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2281 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2341 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2401 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2461 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2521 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2581 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2641 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2701 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2761 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2821 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     2881 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2941 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     3001 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3061 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     3121 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     3181 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     3241 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     3301 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     3361 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     3421 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     3481 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     3541 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     3601 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     3661 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     3721 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     3781 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     3841 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3901 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     3961 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4021 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4081 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4141 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4201 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4261 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4321 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4381 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4441 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4501 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4561 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4621 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4681 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4741 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4801 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4861 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4921 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4981 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5041 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5101 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5161 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5221 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5281 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5341 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5401 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5461 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5521 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5581 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5641 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5701 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5761 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5821 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5881 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5941 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6001 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6061 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6121 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6181 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6241 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
