LOCUS       sequence-495673-        7652 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  Constructs used for LplA-catalyzed labeling in mammalian cells. LplA
            is expressed as a fusion with mCherry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7652)
  TITLE     Ting Lab LplA Plasmids 2025
REFERENCE   2  (bases 1 to 7652)
  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: His6-mCherry-Flag-LplA W37V and E265R
FEATURES             Location/Qualifiers
     source          1..7652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      63..491
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(150..169)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     360..381
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(500..520)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        505..834
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      685..820
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     747..766
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             901..1695
                     /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(955..974)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1565..1584
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1869..1990
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2039..2055)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2039..2055)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2052..2074)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2063..2079
                     /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(2087..2093,2094..2111,2112..2117))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2132..2153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2270..2287)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2441..3029)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2521..2540)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3200..3991,3992..4060))
                     /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     3823..3842
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4061..4165)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4240..4259)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4431..4810
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4811..5014
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4965..4985
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5059..5078
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5059..5077
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      5097..5106
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5118..5135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5148..5855
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5293..5311)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5561..5580
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             5865..5888
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    6928..7516
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6981..7001)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7399..7410)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7525..7646
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
       61 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
      121 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
      181 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
      241 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
      301 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
      361 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
      421 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
      481 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
      541 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
      601 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
      661 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
      721 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
      781 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
      841 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
      901 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
      961 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     1021 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     1081 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     1141 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     1201 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     1261 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     1321 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     1381 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     1441 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     1501 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     1561 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     1621 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     1681 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     1741 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     1801 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     1861 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     1921 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     1981 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     2041 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     2101 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     2161 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     2221 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2281 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2341 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     2401 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2461 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2521 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2581 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2641 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2701 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2761 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2821 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2881 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     2941 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3001 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3061 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3121 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3181 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3241 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3301 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3361 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3421 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3481 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3541 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3601 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3661 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3721 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     3781 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3841 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     3901 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     3961 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4021 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4081 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4141 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     4201 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     4261 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     4321 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     4381 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
     4441 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     4501 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     4561 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     4621 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     4681 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     4741 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     4801 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     4861 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     4921 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     4981 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc
     5041 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt gcggccgcca
     5101 ccatggtgcg gggttctcat catcatcatc atcatggtat ggctagcatg gtgagcaagg
     5161 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     5221 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     5281 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     5341 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     5401 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     5461 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     5521 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     5581 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     5641 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     5701 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     5761 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     5821 gccactccac cggcggcatg gacgagctgt acaagaagga tccagactac aaggatgacg
     5881 acgataagat gagcaccctg agactgctga tcagcgacag ctacgacccc tggttcaacc
     5941 tggccgtgga ggagtgcatc ttcagacaga tgcccgccac ccagagagtg ctgttcctgg
     6001 tgagaaacgc cgacaccgtg gtgatcggca gagcccagaa cccctggaag gagtgcaaca
     6061 ccagaagaat ggaggaggac aacgtgagac tggccagaag aagcagcggc ggcggcgccg
     6121 tgttccacga cctgggcaac acctgcttca ccttcatggc cggcaagccc gagtacgaca
     6181 agaccatcag caccagcatc gtgctgaacg ccctgaacgc cctgggcgtg agcgccgagg
     6241 ccagcggcag aaacgacctg gtggtgaaga ccgtggaggg cgacagaaag gtgagcggca
     6301 gcgcctacag agagaccaag gacagaggct tccaccacgg caccctgctg ctgaacgccg
     6361 acctgagcag actggccaac tacctgaacc ccgacaagaa gaagctggcc gccaagggca
     6421 tcaccagcgt gagaagcaga gtgaccaacc tgaccgagct gctgcccggc atcacccacg
     6481 agcaggtgtg cgaggccatc accgaggcct tcttcgccca ctacggcgag agagtggagg
     6541 ccgagatcat cagccccaac aagacccccg acctgcccaa cttcgccgag accttcgcca
     6601 gacagagcag ctgggagtgg aacttcggcc aggcccccgc cttcagccac ctgctggacg
     6661 agagattcac ctggggcggc gtgagactgc acttcgacgt ggagaagggc cacatcacca
     6721 gagcccaggt gttcaccgac agcctgaacc ccgcccccct ggaggccctg gccggcagac
     6781 tgcagggctg cctgtacaga gccgacatgc tgcagcagga gtgcgaggcc ctgctggtgg
     6841 acttccccga gcaggagaag gagctgagag agctgagcgc ctggatggcc ggcgccgtga
     6901 gataagaatt ctgcagatat ctcgcgaaat caacctctgg attacaaaat ttgtgaaaga
     6961 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     7021 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     7081 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     7141 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     7201 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     7261 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     7321 aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg
     7381 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     7441 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     7501 tgggccgcct ccccgcttaa ttaaaacttg tttattgcag cttataatgg ttacaaataa
     7561 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     7621 ttgtccaaac tcatcaatgt atcttacaat tg
//
