LOCUS       sequence-495665-        6458 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  Plasmid used to express E2p, a site-specific acceptor protein for 
            LplA, in HEK293T cells. Used to evaluate specific labeling activity 
            of LplA variants..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6458)
  TITLE     Ting Lab LplA Plasmids 2025
REFERENCE   2  (bases 1 to 6458)
  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-HA-E2p-YFP
FEATURES             Location/Qualifiers
     source          1..6458
                     /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             952..978
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1252..1254,1255..1257,1258..1968)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1297..1318)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1558..1577)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1905..1926
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2011..2029)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2012..2029)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2049..2066)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2055..2279
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2325..2753
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2412..2431)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2622..2643
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2762..2782)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2767..3096
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2947..3082
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3009..3028
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3163..3957
                     /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(3217..3236)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3827..3846
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4131..4252
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4168..4187)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4222..4241
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4301..4317)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4301..4317)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4314..4336)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4325..4341
                     /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(4349..4355,4356..4373,4374..4379))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4394..4415
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4532..4549)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4703..5291)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4783..4802)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5462..6253,6254..6322))
                     /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     6085..6104
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6323..6427)
                     /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 ctatccgtac
      961 gacgtaccag actacgcaat ggctatcgaa atcaaagtac cggacatcgg ggctgatgaa
     1021 gttgaaatca ccgagatcct ggtcaaagtg ggcgacaaag ttgaagccga acagtcgctg
     1081 atcaccgtag aaggcgacaa agcttctatg gaagttccgg cgccgtttgc aggcgtcgtg
     1141 aaggaactga aagtcaacgt tggcgataaa gtgaaaactg gctcgctgat tatgatcttc
     1201 gaagttgaag gcgcagcgcc tgcggcagct cctgcgaagg atccgagctc gatggtgagc
     1261 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1321 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1381 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1441 accttcggct acggcctgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac
     1501 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1561 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1621 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1681 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1741 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1801 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1861 taccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1921 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tatacaagga attctgaaaa
     1981 aaactcgagc atgcatctag agggccctat tctatagtgt cacctaaatg ctagagctcg
     2041 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2101 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2161 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2221 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2281 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2341 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2401 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2461 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2521 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2581 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2641 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2701 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     2761 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2821 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     2881 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     2941 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3001 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3061 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3121 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3181 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3241 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3301 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     3361 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3421 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     3481 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3541 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3601 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     3661 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     3721 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     3781 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     3841 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     3901 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg
     3961 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg
     4021 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4081 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4141 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4201 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4261 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4321 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4381 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4441 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4501 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4561 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4621 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4681 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4741 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4801 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4861 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4921 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4981 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5041 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5101 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5161 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5221 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5281 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5341 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5401 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5461 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5521 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5581 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5641 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5701 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5761 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5821 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5881 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5941 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6001 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6061 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6121 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6181 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6241 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6301 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6361 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6421 ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
