LOCUS       sequence-443020-        6981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expression of HaloTags to outer surface in mammalian cells 
            coupled with a EGFP transfection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6981)
  TITLE     Frank Lab Plasmids 2024
REFERENCE   2  (bases 1 to 6981)
  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"
COMMENT     Sequence Label: CMV-pDisplay-Halo-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..6981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(4..57)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(76..222)
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     CDS             complement(226..255)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(262..1146)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             complement(1174..1200)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     sig_peptide     complement(1204..1263)
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     primer_bind     complement(1343..1362)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1344..1362)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1407..1610)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1436..1456)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1611..1990
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(2002..2457)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2139..2160)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2351..2370
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2484..2588
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2589..2657,2658..3449)
                     /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     complement(2807..2826)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     enhancer        3509..3700
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3532..3552)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     3779..3798
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3897..4691
                     /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             3897..4691
                     /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(3951..3970)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4561..4580
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4879..4898)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4923..4970
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5299..5887
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5788..5807
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    6019..6243
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6232..6249
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(6268..6981,1..3))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(6271..6981,1..3))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6313..6334)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6662..6681
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6921..6942
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 cactgggcca ggattctcct cgacgtcacc gcatgttagc agacttcctc tgccctctcc
       61 actgccgcgg ccaccacgtg gcttcttctg ccaaagcatg atgaggatga taagggagat
      121 gatggtgagc accaccaggg ccaggatggc tgagatcacc accaccttaa agggcaagga
      181 gtgtggcacc acgatgacct cctgcgtgtc ctggcccaca gcattcagat cctcttctga
      241 gatgagtttt tgttcgtcga caccggaaat ctccagagta gacagccagc gcgcgatctc
      301 gctgccgatc aggtccgggt tgtcttcttg cagcagattc agacccgggc cgatgtccac
      361 agccttgcag ttaggcaggc ttttggccag gcgagcggct tcggccggtg ggatcagaac
      421 gcctggggtg ccccagaaca gcagcttcgg gacaggggac tggtgcagcc agtccatgta
      481 ttcttcgacc agcgcgacga tgttcgctgg ctcaccggcg attggcagct cgtttgggaa
      541 gcgccacagt ggctcgcggt caacaggatt caggaacggc tcgcggtaat ggtccatctc
      601 gacttcagtc agcgggcgga cgacacccat cggcagcgta ccctcgataa aaacgttctg
      661 atcgatgatc agcttgcggc cgacgtcggt ggtgcggaag gcctggaagg tctcgcgggc
      721 aaattctggc cattcgtccc aggtcgggat agggcggatg aactccataa atgcaatacc
      781 tttgacgcgc tctggattgc gcttggccca gtggaaaccc agagcggagc cccagtcgtg
      841 aatgaccagg acgacctctt ccagacccag ggcttcgatg aaggcatcca tgaagcggac
      901 gtggtcgtcg aagaaataac ccaggtctgg tttgtcggat ttgcccatac cgatcaggtc
      961 tggagcaatg cagcgatggg tcggtgcaac atgcgggatg atgttgcgcc acacgtagga
     1021 ggaggtcggg ttaccgtgca ggaacagcac aggggtgcca tcgcgcggac caacatcgac
     1081 gtagtgcatg cgctcgccca ggacttccac ataatggggg tcgaatggaa agccagtacc
     1141 gatttcggat cctgatctgg ccggctgggc cccagcataa tctggaacat catatggata
     1201 gtcaccagtg gaacctggaa cccagagcag cagtacccat agcaggagtg tgtctgtctc
     1261 catggtggat atccccaagc cgaattccag cacactggcg gccgttacta gtggatccga
     1321 gctcggtacc aagcttgggt ctccctatag tgagtcgtat taatttcgat aagccagtaa
     1381 gcagtgggtt ctctagttag ccagagagct ctgcttatat agacctccca ccgtacacgc
     1441 ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga
     1501 ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt
     1561 gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata
     1621 gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg
     1681 gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata
     1741 tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt
     1801 caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg
     1861 cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac
     1921 tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat
     1981 aatcaatgtc aacgcgcgcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     2041 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     2101 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     2161 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     2221 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     2281 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     2341 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     2401 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     2461 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2521 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2581 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2641 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2701 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2761 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2821 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2881 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2941 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3001 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     3061 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3121 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3181 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3241 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3301 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3361 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3421 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3481 agattgattt attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     3541 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3601 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3661 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3721 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3781 tttatgcaga ggccgaggcc cggcctctga gctattccag aagtagtgag gaggcttttt
     3841 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     3901 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     3961 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     4021 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     4081 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4141 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4201 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4261 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4321 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4381 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     4441 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     4501 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     4561 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     4621 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     4681 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     4741 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4801 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4861 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     4921 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     4981 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     5041 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     5101 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     5161 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5221 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5281 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5341 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5401 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5461 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5521 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5581 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5641 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5701 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5761 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5821 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5881 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5941 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccactgg
     6001 ttctttccgc ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt
     6061 cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac
     6121 tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt
     6181 ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca
     6241 cagtcgaggc tgatctcgag cggccgccta cttgtacagc tcgtccatgc cgagagtgat
     6301 cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt
     6361 gctcagggcg gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc
     6421 gatgggggtg ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg
     6481 gcggatcttg aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt
     6541 gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc
     6601 gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg
     6661 ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg
     6721 catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg
     6781 cacgccgtag gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt
     6841 gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac
     6901 gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt
     6961 gaacagctcc tcgcccttgc t
//
