LOCUS       sequence-475100-        6450 bp    DNA     circular SYN 01-JUL-2025
DEFINITION  Express HiBiT-IRES Fusion Protein in Mammalian Cells under a TK 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6450)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 6450)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pBiT4.2-N [HiBiT-IRES-luc2/TK/Blast] Vector
FEATURES             Location/Qualifiers
     source          1..6450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        222..973
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     misc_feature    1213..1785
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1236..1787
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1380..1397)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1607..1626
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1787..3439
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    3513..3634
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3524..3543)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3749..3782)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3792..3839
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3871..4665
                     /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(3925..3944)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4535..4554
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4823..5411)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4903..4922)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5537..5585
                     /note="synthetic polyadenylation signal"
     CDS             complement(5609..6007)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6004..6013
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6038..6395)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6052..6187
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6204..6395
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6352..6372
                     /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 aaatgagtct tcggacctcg
      241 cgggggccgc ttaagcggtg gttagggttt gtctgacgcg gggggagggg gaaggaacga
      301 aacactctca ttcggaggcg gctcggggtt tggtcttggt ggccacgggc acgcagaaga
      361 gcgccgcgat cctcttaagc acccccccgc cctccgtgga ggcgggggtt tggtcggcgg
      421 gtggtaactg gcgggccgct gactcgggcg ggtcgcgcgc cccagagtgt gaccttttcg
      481 gtctgctcgc agacccccgg gcggcgccgc cgcggcggcg acgggctcgc tgggtcctag
      541 gctccatggg gaccgtatac gtggacaggc tctggagcat ccgcacgact gcggtgatat
      601 taccggagac cttctgcggg acgagccggg tcacgcggct gacgcggagc gtccgttggg
      661 cgacaaacac caggacgggg cacaggtaca ctatcttgtc acccggaggc gcgagggact
      721 gcaggagctt cagggagtgg cgcagctgct tcatccccgt ggcccgttgc tcgcgtttgc
      781 tggcggtgtc cccggaagaa atatatttgc atgtctttag ttctatgatg acacaaaccc
      841 cgcccagcgt cttgtcattg gcgaagtcga acacgcagat gcagtcgggg cggcgcggtc
      901 ccaggtccac ttcgcatatt aaggtgacgc gtgtggcctc gaacaccgag cgaccctgca
      961 gcgacccgct taaaagcttg gcaatccggt actgttggta aagccaccgc tagcgatcgc
     1021 catggtgagc ggctggcggc tgttcaagaa gattagcggg agctccggtg gctcgagcgg
     1081 tgggaattct ggtggaggat ccggtggagc tagcggaaga tctggttcta gaggtcaagc
     1141 tttgaccgac caagcgacgc ccaacctgcc atcacggacc acaacggttt ccctctagcg
     1201 ggatcaattc cgcccctctc cctccctccc ccctaacgtt actggccgaa gccgcttgga
     1261 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
     1321 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     1381 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     1441 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     1501 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     1561 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     1621 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     1681 ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc
     1741 ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg aagatgccaa
     1801 aaacattaag aagggcccag cgccattcta cccactcgaa gacgggaccg ccggcgagca
     1861 gctgcacaaa gccatgaagc gctacgccct ggtgcccggc accatcgcct ttaccgacgc
     1921 acatatcgag gtggacatta cctacgccga gtacttcgag atgagcgttc ggctggcaga
     1981 agctatgaag cgctatgggc tgaatacaaa ccatcggatc gtggtgtgca gcgagaatag
     2041 cttgcagttc ttcatgcccg tgttgggtgc cctgttcatc ggtgtggctg tggccccagc
     2101 taacgacatc tacaacgagc gcgagctgct gaacagcatg ggcatcagcc agcccaccgt
     2161 cgtattcgtg agcaagaaag ggctgcaaaa gatcctcaac gtgcaaaaga agctaccgat
     2221 catacaaaag atcatcatca tggatagcaa gaccgactac cagggcttcc aaagcatgta
     2281 caccttcgtg acttcccatt tgccacccgg cttcaacgag tacgacttcg tgcccgagag
     2341 cttcgaccgg gacaaaacca tcgccctgat catgaacagt agtggcagta ccggattgcc
     2401 caagggcgta gccctaccgc accgcaccgc ttgtgtccga ttcagtcatg cccgcgaccc
     2461 catcttcggc aaccagatca tccccgacac cgctatcctc agcgtggtgc catttcacca
     2521 cggcttcggc atgttcacca cgctgggcta cttgatctgc ggctttcggg tcgtgctcat
     2581 gtaccgcttc gaggaggagc tattcttgcg cagcttgcaa gactataaga ttcaatctgc
     2641 cctgctggtg cccacactat ttagcttctt cgctaagagc actctcatcg acaagtacga
     2701 cctaagcaac ttgcacgaga tcgccagcgg cggggcgccg ctcagcaagg aggtaggtga
     2761 ggccgtggcc aaacgcttcc acctaccagg catccgccag ggctacggcc tgacagaaac
     2821 aaccagcgcc attctgatca cccccgaagg ggacgacaag cctggcgcag taggcaaggt
     2881 ggtgcccttc ttcgaggcta aggtggtgga cttggacacc ggtaagacac tgggtgtgaa
     2941 ccagcgcggc gagctgtgcg tccgtggccc catgatcatg agcggctacg ttaacaaccc
     3001 cgaggctaca aacgctctca tcgacaagga cggctggctg cacagcggcg acatcgccta
     3061 ctgggacgag gacgagcact tcttcatcgt ggaccggctg aagagcctga tcaaatacaa
     3121 gggctaccag gtagccccag ccgaactgga gagcatcctg ctgcaacacc ccaacatctt
     3181 cgacgccggg gtcgccggcc tgcccgacga cgatgccggc gagctgcccg ccgcagtcgt
     3241 cgtgctggaa cacggtaaaa ccatgaccga gaaggagatc gtggactatg tggccagcca
     3301 ggttacaacc gccaagaagc tgcgcggtgg tgttgtgttc gtggacgagg tgcctaaagg
     3361 actgaccggc aagttggacg cccgcaagat ccgcgagatt ctcattaagg ccaagaaggg
     3421 cggcaagatc gccgtgtaag tttaaacctg caggcatgca agctgatttg gctgctgcca
     3481 ccgctgagca aggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac
     3541 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     3601 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     3661 gtttcaggtt cagggggaga tgtgggaggt ttttttaagc aagtaaaacc tctacaaatg
     3721 tggtaaaatc ttaattaaga cttctaatat aacttcgtat agcatacatt atacgaagtt
     3781 atggttccac tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca
     3841 aggtgaggaa ctaaacccag gaggcagatc atgattgaac aagatggatt gcacgcaggt
     3901 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     3961 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4021 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4081 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4141 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4201 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4261 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4321 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4381 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4441 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4501 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4561 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4621 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgattcga aatgaccgac
     4681 caagcgacgc ccaaccggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4741 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4801 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4861 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4921 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4981 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5041 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5101 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5161 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5221 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     5281 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5341 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5401 aaggatttca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5461 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5521 tatagtccgg aatcgtcaca caaaaaacca acacacagat gtaatgaaaa taaagatatt
     5581 ttattgcggc catcgtgatg gctagagttt agccctccca cacataacca gagggcagca
     5641 attcacgaat cccaactgcc gtcggctgtc catcactgtc cttcactatg gctttgatcc
     5701 caggatgcag atcgagaagc acctgtcggc accgtccgca ggggctcaag atgcccctgt
     5761 tctcatttcc gatcgcgacg atacaagtca ggttgccagc tgccgcagca gcagcagtgc
     5821 ccagcaccac gagttctgca caaggtcccc cagtaaaatg atatacattg acaccagtga
     5881 agatgcggcc gtcgctagag agagctgcgc tggcgacgct gtagtcttca gagatgggga
     5941 tgctgttgat tgtagccgtt gctctttcaa tgagggtgga ttcttcttga gacaaaggct
     6001 tggccatggt ggcgctagtg tcagaagaat cgagcttttt gcaaaagcct aggcctccaa
     6061 aaaagcctcc tcactacttc tggaatagct cagaggccga ggcggcctcg gcctctgcat
     6121 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg
     6181 gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt
     6241 gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga
     6301 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact
     6361 gacacacatt ccacagctgg ttctttccgc ctcagaaggt acctaaccaa gttcctcttt
     6421 cagaggttat ttcaggccat ggtgctgcgc
//
