LOCUS       sequence-459314-        6809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses cytoplasmic sfGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6809)
  AUTHORS   Jaroenlak P, McCarty KL, Xia B, Lam C, Zwack EE, Yanai I, Bhabha G, 
            Ekiert DC
  TITLE     scRNA-seq reveals transcriptional dynamics of Encephalitozoon 
            intestinalis parasites in human macrophages.
  JOURNAL   bioRxiv [Preprint]. 2024 May 30:2024.05.30.596468. doi: 
            10.1101/2024.05.30.596468.
  PUBMED    38853846
REFERENCE   2  (bases 1 to 6809)
  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"; journalName: "bioRxiv 
            [Preprint]. 2024 May 30:2024.05.30.596468. doi: 
            10.1101/2024.05.30.596468."
COMMENT     Sequence Label: pBEL2838
FEATURES             Location/Qualifiers
     source          1..6809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        47..426
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        427..630
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     581..601
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     627..651
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(778..780,781..1494)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             778..1491
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(808..836)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1431..1451
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     misc_feature    1583..2171
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1636..1656)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2054..2065)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2192..2211)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2236..2284
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2486..2914
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2573..2592)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2783..2804
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2923..2943)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2928..3257
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3108..3243
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3170..3189
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3324..4118
                     /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(3378..3397)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3988..4007
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4294..4415
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4331..4350)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4385..4404
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4464..4480)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4464..4480)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4477..4499)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4488..4504
                     /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(4512..4518,4519..4536,4537..4542))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4557..4578
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4695..4712)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4866..5454)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4946..4965)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5625..6416,6417..6485))
                     /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     6248..6267
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6486..6590)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6665..6684)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
       61 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      121 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      181 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      241 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      301 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      361 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      421 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      481 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      541 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      601 gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga
      661 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccggact
      721 ctagaggatc gaacccttgg atctctagcg aattccctct agacacagac gctcaccatg
      781 agcaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat
      841 gttaatgggc acaaattttc tgtccgtgga gagggtgaag gtgatgctac aaacggaaaa
      901 ctcaccctta aatttatttg cactactgga aaactacctg ttccgtggcc aacacttgtc
      961 actactctga cctatggtgt tcaatgcttt tcccgttatc cggatcacat gaaacggcat
     1021 gactttttca agagtgccat gcccgaaggt tatgtacagg aacgcactat atctttcaaa
     1081 gatgacggga cctacaagac gcgtgctgaa gtcaagtttg aaggtgatac ccttgttaat
     1141 cgtatcgagt taaagggtat tgattttaaa gaagatggaa acattcttgg acacaaactc
     1201 gagtacaact ttaactcaca caatgtatac atcacggcag acaaacaaaa gaatggaatc
     1261 aaagctaact tcaaaattcg ccacaacgtt gaagatggtt ccgttcaact agcagaccat
     1321 tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg
     1381 tcgacacaat ctgtcctttc gaaagatccc aacgaaaagc gtgaccacat ggtccttctt
     1441 gagtttgtaa ctgctgctgg gattacacat ggcatggatg agctctacaa atgagcggcc
     1501 gctcgaggcc ggcaaggccg gatcccccga cctcgacaag ggttcgatcc ctaccggtta
     1561 gtaatgagtt tgatatctcg acaatcaacc tctggattac aaaatttgtg aaagattgac
     1621 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     1681 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     1741 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     1801 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     1861 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     1921 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct
     1981 gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     2041 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     2101 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     2161 cgcctccccg cctggaacgg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     2221 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacgggtgt tgggtcgttt
     2281 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     2341 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     2401 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc agatctgcgc
     2461 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2521 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2581 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2641 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2701 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2761 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2821 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2881 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2941 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3001 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3061 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3121 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3181 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3241 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     3301 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3361 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3421 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3481 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3541 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3601 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3661 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3721 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3781 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3841 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3901 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3961 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4021 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4081 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgcgaa
     4141 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4201 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4261 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4321 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4381 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     4441 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4501 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4561 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4621 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4681 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4741 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4801 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4861 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4921 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4981 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5041 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5101 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5161 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5221 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5281 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5341 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5401 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5461 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5521 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5581 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5641 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5701 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5761 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5821 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5881 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5941 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6001 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6061 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6121 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6181 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6241 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6301 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6361 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6421 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6481 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6541 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6601 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     6661 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     6721 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     6781 acaattgcat gaagaatctg cttagggtt
//
