LOCUS       sequence-473309-        7598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express ZNRF3-LgBiT 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 7598)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7598)
  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: 
            "Unpublished"
COMMENT     Sequence Label: ZNRF3-LgBiT, BiBiT-Ready Vector
FEATURES             Location/Qualifiers
     source          1..7598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    17..98
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     42..61
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(3652..3670)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3653..3670)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        3790..4093
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4248..4268
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        4301..4348
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4380..5174
                     /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(4434..4453)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5044..5063
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(5332..5920)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5412..5431)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    6046..6094
                     /note="synthetic polyadenylation signal"
     CDS             complement(6118..7155)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             6131..6139
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     promoter        complement(7186..7543)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7200..7335
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        7352..7543
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     7500..7520
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtg acgtcggcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
       61 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
      121 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
      181 aatgtggtat ggctgattat gatccgcggc cgcaatacgt cgacgttatc agctgacttc
      241 gtacgagagc ctaggtgttt ctagagtcgc ggccttagct gttgatggtt actcggaaca
      301 gcatggagcc gtcgggggtg atcaggcgct cgtcgataat tttgttgccg ttccacaggg
      361 tccctgttac agtgatcttt ttgccgtcga acacggcgat gccttcatac ggccgtccga
      421 aatagttcag catgttcggc gtaaccccgt cgattaccag tgtgccatag ggcaggatca
      481 ccttaaagtg atgatcatcc acagggtaca ccaccttaaa cacctcttcg atctgggcca
      541 tttggtcggc gctcagacct tcatacggga tgatgacatg gatgtcgatc ttcagggcat
      601 tttcaccgct ccggacaatc ctttggatcg gagttacgga cacggcgaga ttctgcagca
      661 aactggacac acctccctgt tcaaggactt ggtccaggtt gtaggcggct gtctgttccc
      721 agtccccaac gaaatcttcg agtgtgaaga caccgctcga gcctccacct ccgctcccgc
      781 caccacctga actgccttga gaaacggctc ccgggctgct gctgtctgct gagtgagatc
      841 tcggtcctgc agcctcagtg gcagtggtgc tggactcctg agtgtcctgg agggcactag
      901 ggaagttggc cctgacagca cccgcctcct ccggagggca gccaggccgc agtagggccc
      961 tagcctggca gcacagagcc cgctcctctt cccgggttgc tcccaggccc ctgtggggcc
     1021 gtggggtatc cgggcctcgc gtcccacccc aggagccgag gctgtgggtc ccttggcagt
     1081 ccgagggcag gcccagatca cagtccacat cctctggaat gatggggatg cgctggctca
     1141 ggtcccgggc cccggggtag cagccgggcg gtggttcctc ggtgagcgtg tactgcacac
     1201 tcaccgagta gtcctcagtg tagcagccgc tgccccctcc gcccccgcgg gccacctgct
     1261 tctcttcata gaagcagcag ggcagaccct cgtatttcac cccatctgtc ctgggcaaat
     1321 ggtcggggtg aaggccgtac aagccctggg agcttcctga ctggggctcc ccacccgccg
     1381 ggccagagcc ctcgtagagg tggggcccca ggaacaaggt gctgctgcca gctgctccgg
     1441 cgctaggccc ggctggggag ggctggggct cgcagcagca ggcacacgac ggcaactcgt
     1501 ggcccccctt ccaggtcctc ctgaggtcag gggcggcccc aggagaagcc tcgagcccca
     1561 cttctgaggt agagctattg ggccccctgt gctccaggga ggagttgctg ctgtagttca
     1621 tctccaggct gcaggtggac acctgatccc ccgagggaga ggcagggccc ggccaaggct
     1681 cgccccgccc agcaccatga ctgtgcaccg ccgggagctc ctcgggaggc ggggagccct
     1741 cgaagcacag ggcaggtccc cggcccgagc tgcccgagcc ccccgcctca ctggcacgac
     1801 aggggctccg gctgcggtag atgaaggggt catagtcgct gctgagggag ctgcggaagg
     1861 tggagcagct cccgtacacg ccctggttgc tgacctcagt gcagtctacc acagagtcac
     1921 tggaggaaca gtggcactgg ccggagctgc tgctgctgct gctgtcgctg cctgggcagt
     1981 cggccaggta gccactgcac accgagcggt ggccgtgata gcagctgaag ctggacgtgc
     2041 tgccgctctc caggtgggag ggcggggcca cgtgcaccac ggtggggaag agcaggctgc
     2101 cactgccgct cggaggaaag gcacgggccg ggccccgggg tgcgaggcta ggtggggact
     2161 gcccctcctg ctccgggtag ctgaggccct ggaagtagta gtgctggtac atggtctcat
     2221 actgggagaa gcaagctgcc ttggagaagc tgcggccact caacttgggc ctgcggaagg
     2281 ggtgggctgg ggagtaggcc cggtgctcca ggccgcagcg gtgagcggcc aggctgtggt
     2341 ccaggtggag gggtgggtag ctgcggatgt aggcgggggt ctgcggggaa tagaggctct
     2401 gctccccgtg ccggtccatg gtcagcaagg tgacggggtt gccgtgggag tccatgcttg
     2461 tcctcgtagg gtaggctggg atggcgttgg tcctgtgcac gcggccgggg taatgcaccg
     2521 gcagggtcac cctctgctgc cgaccacgtg agaggttgct ggtctccaca cacaccgcgc
     2581 ttgggtttcc cttttgttct atgatgttgt gccgacagtg ggggcaggtg tggtgctgca
     2641 gcagccaggg gtccacgcac ttcctgtgaa accggtgagt acaggggatg acccgcagct
     2701 cctctccatc aatgtacttc tccagacaga tggcacagtc ggacgtggag ctgctgctga
     2761 gtgtgtccag ggccccacag ctcccctccc ggcgcccctt gctcttggag ttgaactttc
     2821 tggtttccat cttctctaga gcctgcacag ccagcctgtt catggaattc tgactgcgtc
     2881 gctgcttcag cttgattttg acaaggagga tgaggcagac caaggagacc acgacgaaga
     2941 aagccaggaa aatccccatg tcaaagtatt cagtgggttg tcgaggaggg cggtgctgga
     3001 tccttgctcg agccactttc tgcttgttga cgatgttcat cagcttaatg gcatctgcac
     3061 ccttcacata caccaccggc ctcttgagcg ggtcttcaga gccctggttc agctgatcaa
     3121 tagcttctgg gttttcagac acatcaaaga tgactgcagt agctccccgc tgtactgctc
     3181 gcttggcctt gcctaggaca gtgaggcatg gtttcgggtc caattctggc tgttccagct
     3241 tcaccactcc tacccagcca tattcataca agtcctcttc gtcattgtta ttacataggc
     3301 ccagtgggtg catctgcacg atctcgccct cggcgctgag cgtggccccg gcccgcgaga
     3361 agcggcccgt gaggccggtg gtgtaggtgg tgtaatcgcc gcttgggctc gactcgaaca
     3421 gcaccacctc cacgaacgcc gtctccttgg cccgcgccgc gccgggcccc gcggccgcca
     3481 gcagcagccc gagcagcagc ggcagcggcg gcggcggcgg caggcggctg caccggaggc
     3541 cgcgggggcg gcggcgcagg cggcggcggc ggcggcccgt ggcccctggg cgcccgcccg
     3601 agcgcggcct catggcgatc gctagctgca aaaagaacaa gtagcttgta ttctatagtg
     3661 tcacctaaat gggcgatctg acggttcact aaaccagctc tgcttatata ggcctccctg
     3721 gcgcgccact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     3781 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     3841 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     3901 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     3961 tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     4021 tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg tattagtcat
     4081 cgctattacc atggtgatgc ggttttggca gtacaccaat gggcgtggat agcggtttga
     4141 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     4201 aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg
     4261 taggcgtgta cggtgggagg tcaaatcgaa ttctaatact gttgacaatt aatcatcggc
     4321 atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaacccagg aggcagatca
     4381 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4441 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4501 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4561 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4621 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4681 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4741 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4801 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4861 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     4921 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4981 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     5041 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     5101 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     5161 acgagttctt ctgattcgaa atgaccgacc aagcgacgcc caaccggtat cagctcactc
     5221 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5281 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5341 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5401 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5461 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5521 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5581 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5641 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5701 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5761 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5821 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5881 ttgcaagcag cagattacgc gcagaaaaaa aggatttcaa gaagatcctt tgatcttttc
     5941 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6001 atcaaaaagg atcttcacct agatcctttt atagtccgga atcgtcacac aaaaaaccaa
     6061 cacacagatg taatgaaaat aaagatattt tattgcggcc atcgtgatgg ctagagtcta
     6121 aactcgacct acctccttag cgcgcggtcg tgtactgggc cggcggttgc cgctgtcggc
     6181 cagcacctcg acgcagccgt cggtccatac ggctgcgctc cggcgagcga tttgtgtgcg
     6241 accgacggtg ccggccccgc tgcggacgat ggcatcgcag cggccttgag cccaggcagc
     6301 atcgtcgaag ttgccgtcca cgaggctctg gtagagctga tccaggccga tgcgcagcat
     6361 gtaggctcgc agacgagggc tgccggccag ctcgggatgc cggcgctcga agtagcgagt
     6421 ctgctgctcc atgcaagcca gccagggccg ccagaagaag atgttggcca cctcgtactg
     6481 actgtccccg aacatagctt cggaccagtc gattacggcg gtgatgcggc cgttgtcggt
     6541 caggacgttg ttgctgccga agtcggcatg gacgaggtgg cgcacctcgg gacagtcttc
     6601 ggcccacagc atcagttcgt ccagggcttg agctacgctg gcggacacgg tgtcgtccat
     6661 cacggtctgc cagtggtaga catggggatc agcaatggcg caaatgaaat cccgccaggt
     6721 tgtgtactgg ccgatgcctt ggggcccgaa tgggccgaag ccgctggttt gactgaggtc
     6781 ggcggcggca atagcatcca tagcttcggc gacaggctgt aacacagcag gcagctctgt
     6841 ttcggggagg tcttggagag taacgccttg ggcgcgtcta ctgatgcagt atgtcaggct
     6901 ctcgctgaac tcgccgatgt ccaacacttc ggggatgggt agtgcagcgc tggcgaagtg
     6961 gcggtacacg tagcggtctt tgtagaagcc atcagcgcag ctattcaccc gcagtacata
     7021 gccgcgtccg ccgacatcga agctgaaggc tcggctctct tcgccctccg acaactgcat
     7081 caggtcgctc acactgtcga acttctcgat gagaaatttt tcaacgctgg tagcggtgag
     7141 ttcgggcttc ttcatggtgg cgctagtgtc agaagaatcg agctttttgc aaaagcctag
     7201 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc
     7261 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     7321 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     7381 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga
     7441 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     7501 ccctaactga cacacattcc acagctggtt ctttccgcct cagaaggtac ctaaccaagt
     7561 tcctctttca gaggttattt caggccatgg tgctgcgc
//
