LOCUS       sequence-441428-        6926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a putative signal peptide from Encephalitozoon 
            intestinalis protein Eint_060140 fused to GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6926)
  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 2024.05.30.596468
REFERENCE   2  (bases 1 to 6926)
  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 
            2024.05.30.596468"
COMMENT     Sequence Label: pBEL2999
FEATURES             Location/Qualifiers
     source          1..6926
                     /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(892..894,895..897,898..1611)
                     /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="MASKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             892..1608
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(925..953)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1548..1568
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     misc_feature    1700..2288
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1753..1773)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2171..2182)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2309..2328)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2353..2401
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2603..3031
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2690..2709)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2900..2921
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3040..3060)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3045..3374
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3225..3360
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3287..3306
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3441..4235
                     /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(3495..3514)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4105..4124
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4411..4532
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4448..4467)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4502..4521
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4581..4597)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4581..4597)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4594..4616)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4605..4621
                     /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(4629..4635,4636..4653,4654..4659))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4674..4695
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4812..4829)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4983..5571)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5063..5082)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5742..6533,6534..6602))
                     /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     6365..6384
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6603..6707)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6782..6801)
                     /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 ttgttacttc tctcagcagt tgcttttgtt agcgctacag cagtccagtc aggtgttgtc
      841 tcccagccta caacacccat tccgattctt cctggacagc cgatgggggg catggccagc
      901 aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt
      961 aatgggcaca aattttctgt ccgtggagag ggtgaaggtg atgctacaaa cggaaaactc
     1021 acccttaaat ttatttgcac tactggaaaa ctacctgttc cgtggccaac acttgtcact
     1081 actctgacct atggtgttca atgcttttcc cgttatccgg atcacatgaa acggcatgac
     1141 tttttcaaga gtgccatgcc cgaaggttat gtacaggaac gcactatatc tttcaaagat
     1201 gacgggacct acaagacgcg tgctgaagtc aagtttgaag gtgataccct tgttaatcgt
     1261 atcgagttaa agggtattga ttttaaagaa gatggaaaca ttcttggaca caaactcgag
     1321 tacaacttta actcacacaa tgtatacatc acggcagaca aacaaaagaa tggaatcaaa
     1381 gctaacttca aaattcgcca caacgttgaa gatggttccg ttcaactagc agaccattat
     1441 caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca ttacctgtcg
     1501 acacaatctg tcctttcgaa agatcccaac gaaaagcgtg accacatggt ccttcttgag
     1561 tttgtaactg ctgctgggat tacacatggc atggatgagc tctacaaatg agcggccgct
     1621 cgaggccggc aaggccggat cccccgacct cgacaagggt tcgatcccta ccggttagta
     1681 atgagtttga tatctcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     1741 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     1801 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     1861 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     1921 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     1981 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     2041 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac
     2101 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     2161 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     2221 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     2281 ctccccgcct ggaacggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     2341 cccgcgctat gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt
     2401 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     2461 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     2521 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc
     2581 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2641 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2701 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2761 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2821 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2881 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2941 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3001 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3061 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3121 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3181 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3241 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3301 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3361 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     3421 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3481 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3541 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3601 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     3661 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3721 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3781 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3841 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     3901 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     3961 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4021 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4081 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4141 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4201 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgcgaaatg
     4261 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4321 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4381 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4441 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4501 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4561 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4621 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4681 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4741 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4801 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4861 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4921 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4981 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5041 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5101 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5161 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5221 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5281 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5341 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5401 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5461 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5521 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5581 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5641 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5701 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5761 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5821 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5881 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5941 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6001 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6061 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6121 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6181 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6241 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6301 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6361 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6421 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6481 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6541 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6601 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6661 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6721 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     6781 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     6841 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     6901 attgcatgaa gaatctgctt agggtt
//
