LOCUS       sequence-479772-        5517 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express CISH-NanoLuc 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 5517)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5517)
  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            "Unpublished"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: CISH-NanoLuc Fusion Vector
FEATURES             Location/Qualifiers
     source          1..5517
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1860..2372
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(2460..2478)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2515..2636
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2526..2545)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2580..2599
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2881..3238
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2881..3072
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2904..2924)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3089..3224
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3253..3300
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3332..4126
                     /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(3386..3405)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3996..4015
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4190..4238
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(4477..5065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4557..4576)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    5181..5464
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcgat cgccatggtt ctctgcgttc
     1081 agggacctcg tcctttgctg gctgttgagc ggactggtca gcggccactg tgggctccgt
     1141 ccctggaact gcccaagcca gtcatgcagc cattgcctgc tggtgccttc ctcgaagagg
     1201 ttgcagaggg taccccagcc cagacagaga gtgagccaaa ggtgctggac ccagaggagg
     1261 atctgctgtg catagccaag accttctcct accttcggga atctggctgg tattggggtt
     1321 ccattacggc cagcgaggcc cgacaacacc tgcagaagat gccagaaggc acgttcttag
     1381 tacgtgacag cacgcacccc agctacctgt tcacgctgtc agtgaaaacc actcgtggcc
     1441 caaccaatgt acgcattgag tatgccgact ccagcttccg tctggactcc aactgcttgt
     1501 ccaggccacg catcctggcc tttccggatg tggtcagcct tgtgcagcac tatgtggcct
     1561 cctgcactgc tgatacccga agcgacagcc cagatcctgc tcccacccca gccctgccta
     1621 tgcctaagga ggatgcgcct agtgacccag cactgcctgc tcctccacca gctactgctg
     1681 tacacctaaa actggtgcag ccctttgtac gcagaagcag tgcacgcagc ctgcaacacc
     1741 tgtgccgcct tgttatcaac cgtctggtgg ccgatgttga ctgcctgcca ctgccccggc
     1801 gcatggctga ctacctccga cagtacccct tccagctcgt ttctctcggc tcgagcggcg
     1861 tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac aacctggacc
     1921 aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc
     1981 cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc catgtcatca
     2041 tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg
     2101 tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca ctggtaatcg
     2161 acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc atcgccgtgt
     2221 tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg
     2281 agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac ggagtgaccg
     2341 gctggcggct gtgcgaacgc attctggcgt aaggccgcga ctctagagtc gacctgcagg
     2401 catgcaagct gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
     2461 cgctgagcaa taactagcat aaccccttgg ggcggccgct tcgagcagac atgataagat
     2521 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     2581 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     2641 acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag gtttttttaa
     2701 gcaagtaaaa cctctacaaa tgtggtaaaa tcgaatttta acaaaatatt aacgcttaca
     2761 atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgcg
     2821 gatctgcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt
     2881 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2941 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3001 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3061 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3121 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     3181 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     3241 gcttaattaa ctgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     3301 aaggtgagga actaaaccca ggaggcagat catgattgaa caagatggat tgcacgcagg
     3361 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3421 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3481 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     3541 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     3601 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     3661 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     3721 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     3781 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     3841 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     3901 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     3961 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     4021 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     4081 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     4141 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gatggccgca ataaaatatc
     4201 tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagcga taaggatcct
     4261 ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca
     4321 gcaccgtttc tgcggactgg ctttctaccc ggtatcagct cactcaaagg cggtaatacg
     4381 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4441 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4501 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4561 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4621 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4681 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4741 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4801 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4861 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     4921 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4981 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5041 tacgcgcaga aaaaaaggat ttcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5101 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5161 cacctagatc cttttatagt ccggaaatac aggaacgcac gctggatggc ccttcgctgg
     5221 gatggtgaaa ccatgaaaaa tggcagcttc agtggattaa gtgggggtaa tgtggcctgt
     5281 accctctggt tgcataggta ttcatacggt taaaatttat caggcgcgat tgcggcagtt
     5341 tttcgggtgg tttgttgcca tttttacctg tctgctgccg tgatcgcgct gaacgcgttt
     5401 tagcggtgcg tacaattaag ggattatggt aaatccactt actgtctgcc ctcgtagcca
     5461 tcgagataaa ccgcagtact ccggccacga tgcgtccggc gtagaggatc gagatct
//
