LOCUS       sequence-479909-        6000 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express EPOR-SmBiT Fusion Protein in Mammalian Cells under a CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6000)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6000)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: EPOR-SmBiT Vector
FEATURES             Location/Qualifiers
     source          1..6000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        338..641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        642..845
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     796..816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          981..1113
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1096..1115
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1157..1176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1157..1175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2889..3010
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2900..2919)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3125..3158)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3168..3215
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3247..4041
                     /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(3301..3320)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3911..3930
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      4258..4803
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     4704..4723
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4920..4937
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    5087..5135
                     /note="synthetic polyadenylation signal"
     CDS             complement(5159..5557)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5554..5563
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5588..5945)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5602..5737
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5754..5945
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5902..5922
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtt aattaagacg tcggcagtga aaaaaatgct ttatttgtga aatttgtgat
       61 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
      121 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
      181 ctctacaaat gtggtatggc tgattatgat ccgcggccgc aatacgtcga cgttatcagc
      241 tgacttcgta cgagagccta ggattatggc gcgccactag ttattaatag taatcaatta
      301 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      361 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      421 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      481 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
      541 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
      601 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      661 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      721 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
      781 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      841 gagctggttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt tatcacagtt
      901 aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc tgcagaagtt
      961 ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat
     1021 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc
     1081 ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct
     1141 cttaaggcta gagtattaat acgactcact atagggctag cgatcgccat ggaccacctc
     1201 ggggcgtccc tctggcccca ggtcggctcc ctttgtctcc tgctcgctgg ggccgcctgg
     1261 gcgcccccgc ctaacctccc ggaccccaag ttcgagagca aagcggcctt gctggcggcc
     1321 cgggggcccg aagagcttct gtgcttcacc gagcggttgg aggacttggt gtgtttctgg
     1381 gaggaagcgg cgagcgctgg ggtgggcccg ggcaactaca gcttctccta ccagctcgag
     1441 gatgagccat ggaagctgtg tcgcctgcac caggctccca cggctcgtgg tgcggtgcgc
     1501 ttctggtgtt cgctgcctac agccgacacg tcgagcttcg tgcccctaga gttgcgcgtc
     1561 acagcagcct ccggcgctcc gcgatatcac cgtgtcatcc acatcaatga agtagtgctc
     1621 ctagacgccc ccgtggggct ggtggcgcgg ttggctgacg agagcggcca cgtagtgttg
     1681 cgctggctcc cgccgcctga gacacccatg acgtctcaca tccgctacga ggtggacgtc
     1741 tcggccggca acggcgcagg gagcgtacag agggtggaga tcctggaggg ccgcaccgag
     1801 tgtgtgctga gcaacctgcg gggccggacg cgctacacct tcgccgtccg cgcgcgtatg
     1861 gctgagccga gcttcggcgg cttctggagc gcctggtcgg agcctgtgtc gctgctgacg
     1921 cctagcgacc tggaccccct catcctgacg ctctccctca tcctcgtggt catcctggtg
     1981 ctgctgaccg tgctcgcgct gctctcccac cgccgggctc tgaagcagaa gatctggcct
     2041 ggcatcccga gcccagagag cgagtttgaa ggcctcttca ccacccacaa gggtaacttc
     2101 cagctgtggc tgtaccagaa tgatggctgc ctgtggtgga gcccctgcac ccccttcacg
     2161 gaggacccac ctgcttccct ggaagtcctc tcagagcgct gctgggggac gatgcaggca
     2221 gtggagccgg ggacagatga tgagggcccc ctgctggagc cagtgggcag tgagcatgcc
     2281 caggatacct atctggtgct ggacaaatgg ttgctgcccc ggaacccgcc cagtgaggac
     2341 ctcccagggc ctggtggcag tgtggacata gtggccatgg atgaaggctc agaagcatcc
     2401 tcctgctcat ctgctttggc ctcgaagccc agcccagagg gagcctctgc tgccagcttt
     2461 gagtacacta tcctggaccc cagctcccag ctcttgcgtc catggacact gtgccctgag
     2521 ctgcccccta ccccacccca cctaaagtac ctgtaccttg tggtatctga ctctggcatc
     2581 tcaactgact acagctcagg ggactcccag ggagcccaag ggggcttatc cgatggcccc
     2641 tactccaacc cttatgagaa cagccttatc ccagccgctg agcctctgcc ccccagctat
     2701 gtggcttgct ctgtttctca aggcagttca ggtggtggcg ggagcggagg tggaggctcg
     2761 agcggtgtga ccggctaccg gctgttcgag gagattctgt aaggccgcga ctctagagtc
     2821 gacctgcagg catgcaagct gatttggctg ctgccaccgc tgagcaaggc cgcttcgagc
     2881 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     2941 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3001 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg
     3061 ggaggttttt ttaagcaagt aaaacctcta caaatgtggt aaaatcttaa ttaagaattc
     3121 taatataact tcgtatagca tacattatac gaagttatgg atccactgtt gacaattaat
     3181 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa acccaggagg
     3241 cagatcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3301 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3361 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3421 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3481 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3541 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3601 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     3661 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     3721 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     3781 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     3841 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     3901 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     3961 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4021 cttcttgacg agttcttctg attcgaaatg accgaccaag cgacgcccaa ccggtatcag
     4081 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4141 tgtcaaacat gagaattaca acttatatcg tatggggctg acttcaggtg ctacatttga
     4201 agagataaat tgcactgaaa tctagaaata ttttatctga ttaataagat gatcttcttg
     4261 agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac gaaaaaaccg ccttgcaggg
     4321 cggtttttcg aaggttctct gagctaccaa ctctttgaac cgaggtaact ggcttggagg
     4381 agcgcagtca ccaaaacttg tcctttcagt ttagccttaa ccggcgcatg acttcaagac
     4441 taactcctct aaatcaatta ccagtggctg ctgccagtgg tgcttttgca tgtctttccg
     4501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcggactga acggggggtt
     4561 cgtgcataca gtccagcttg gagcgaactg cctacccgga actgagtgtc aggcgtggaa
     4621 tgagacaaac gcggccataa cagcggaatg acaccggtaa accgaaaggc aggaacagga
     4681 gagcgcacga gggagccgcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4741 cgccaccact gatttgagcg tcagatttcg tgatgcttgt caggggggcg gagcctatgg
     4801 aaaaacggct ttgccgcggc cctctcactt ccctgttaag tatcttcctg gcatcttcca
     4861 ggaaatctcc gccccgttcg taagccattt ccgctcgccg cagtcgaacg accgagcgta
     4921 gcgagtcagt gagcgaggaa gcggaatata tcctgtatca catattctgc tgacgcaccg
     4981 gtgcagcctt ttttctcctg ccacatgaag cacttcactg acaccctcat cagtgccaac
     5041 atagtaagcc agtatacact ccgctagcgc tgatgtccgg aatcgtcaca caaaaaacca
     5101 acacacagat gtaatgaaaa taaagatatt ttattgcggc catcgtgatg gctagagttt
     5161 agccctccca cacataacca gagggcagca attcacgaat cccaactgcc gtcggctgtc
     5221 catcactgtc cttcactatg gctttgatcc caggatgcag atcgagaagc acctgtcggc
     5281 accgtccgca ggggctcaag atgcccctgt tctcatttcc gatcgcgacg atacaagtca
     5341 ggttgccagc tgccgcagca gcagcagtgc ccagcaccac gagttctgca caaggtcccc
     5401 cagtaaaatg atatacattg acaccagtga agatgcggcc gtcgctagag agagctgcgc
     5461 tggcgacgct gtagtcttca gagatgggga tgctgttgat tgtagccgtt gctctttcaa
     5521 tgagggtgga ttcttcttga gacaaaggct tggccatggt ggcgctagtg tcagaagaat
     5581 cgagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     5641 cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg
     5701 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
     5761 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     5821 actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     5881 gggagcctgg ggactttcca caccctaact gacacacatt ccacagctgg ttctttccgc
     5941 ctcagaaggt acctaaccaa gttcctcttt cagaggttat ttcaggccat ggtgctgcgc
//
