LOCUS       sequence-471886-        5921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express NCK1-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 5921)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5921)
  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            "Unpublished"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: NCK1-LgBiT Fusion Vector
FEATURES             Location/Qualifiers
     source          1..5921
                     /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(1975..1993)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1976..1993)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        2113..2416
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2571..2591
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        2624..2671
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2703..3497
                     /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(2757..2776)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3367..3386
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3655..4243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3735..3754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4369..4417
                     /note="synthetic polyadenylation signal"
     CDS             complement(4441..5478)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4454..4462
                     /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(5509..5866)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5523..5658
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5675..5866
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5823..5843
                     /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 gaaactgata aatgcttgac aagatataat ttttctcctt
      841 gttcacttgt aaaaattggt gcctttttgt aatgttctac aagttcttcc atggtgctga
      901 atttacgctg cccaatgcag tagacagtct cttttagttg gactttaaaa tgcttgtttt
      961 tcccttgtgc ttttagtgat actgagaaat catttggcga agattcacta tcacgaatga
     1021 ggaaatcccc ttcatgtcct ctttcattta atgccatttc tgcttgatgc ctggtgactt
     1081 tgccataata ccaaggattg ccagcaaact ttccagtgag tgaaggccta atgtaatcac
     1141 actgtggagg tgatggttcc aaacctgaag ttaatggatt attctgcata acggtaacat
     1201 agttttttgg tactagacca accataccat tgatcttcct gcatttccac cactctgggt
     1261 cattttcagg tttttcaata acatccatta catctccttt ctcgaaatta agttcttcat
     1321 cattagatga gctgaatggg taaagagcct gtaccacatg caacacttgc ccagtattta
     1381 ggttattgac gactgctgct aatttctctg acagagaacc cacatggtca cccaaaggac
     1441 tgtcaccttc ttcagttaca tagtttgaag ggaaccatcc aacttgtcca ttgtagctac
     1501 cacgccacca cccatcactg catttctcca tgacgatcac ctttgtcccc tttatcaatg
     1561 ataattcatc ctctctctca gccatgtagt taaatttcac ataagcgggc atgttgaggt
     1621 catagagacg ttcccctggg tcaacaaaac tatcatcagc aggagatgca gaatctggca
     1681 cactaggttt tcttttcact tttccaatgc ctaaggtatc ctttaggttt ttcacaatag
     1741 atgctttccg agcactgttt ttcctttcca catagttaga aggcacaaaa cctgttttat
     1801 tcatggaatt tcgaactcgc caccaggact tagaatcatc cagaagccat aatctctcat
     1861 tcttcttgat gtccaactct tgttcttgtt gggccacata atcaaatttg gctactacca
     1921 ccacttcttc tgccatggcg atcgctagct gcaaaaagaa caagtagctt gtattctata
     1981 gtgtcaccta aatgggcgat ctgacggttc actaaaccag ctctgcttat ataggcctcc
     2041 ctggcgcgcc actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     2101 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     2161 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     2221 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     2281 gtatcatatg ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     2341 ttatgcccag tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt
     2401 catcgctatt accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt
     2461 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     2521 ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg
     2581 cggtaggcgt gtacggtggg aggtcaaatc gaattctaat actgttgaca attaatcatc
     2641 ggcatagtat atcggcatag tataatacga caaggtgagg aactaaaccc aggaggcaga
     2701 tcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     2761 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     2821 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     2881 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     2941 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3001 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3061 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3121 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3181 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     3241 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     3301 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     3361 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     3421 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     3481 ttgacgagtt cttctgattc gaaatgaccg accaagcgac gcccaaccgg tatcagctca
     3541 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3601 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3661 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3721 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3781 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3841 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3901 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3961 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4021 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4081 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4141 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4201 tgtttgcaag cagcagatta cgcgcagaaa aaaaggattt caagaagatc ctttgatctt
     4261 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4321 attatcaaaa aggatcttca cctagatcct tttatagtcc ggaatcgtca cacaaaaaac
     4381 caacacacag atgtaatgaa aataaagata ttttattgcg gccatcgtga tggctagagt
     4441 ctaaactcga cctacctcct tagcgcgcgg tcgtgtactg ggccggcggt tgccgctgtc
     4501 ggccagcacc tcgacgcagc cgtcggtcca tacggctgcg ctccggcgag cgatttgtgt
     4561 gcgaccgacg gtgccggccc cgctgcggac gatggcatcg cagcggcctt gagcccaggc
     4621 agcatcgtcg aagttgccgt ccacgaggct ctggtagagc tgatccaggc cgatgcgcag
     4681 catgtaggct cgcagacgag ggctgccggc cagctcggga tgccggcgct cgaagtagcg
     4741 agtctgctgc tccatgcaag ccagccaggg ccgccagaag aagatgttgg ccacctcgta
     4801 ctgactgtcc ccgaacatag cttcggacca gtcgattacg gcggtgatgc ggccgttgtc
     4861 ggtcaggacg ttgttgctgc cgaagtcggc atggacgagg tggcgcacct cgggacagtc
     4921 ttcggcccac agcatcagtt cgtccagggc ttgagctacg ctggcggaca cggtgtcgtc
     4981 catcacggtc tgccagtggt agacatgggg atcagcaatg gcgcaaatga aatcccgcca
     5041 ggttgtgtac tggccgatgc cttggggccc gaatgggccg aagccgctgg tttgactgag
     5101 gtcggcggcg gcaatagcat ccatagcttc ggcgacaggc tgtaacacag caggcagctc
     5161 tgtttcgggg aggtcttgga gagtaacgcc ttgggcgcgt ctactgatgc agtatgtcag
     5221 gctctcgctg aactcgccga tgtccaacac ttcggggatg ggtagtgcag cgctggcgaa
     5281 gtggcggtac acgtagcggt ctttgtagaa gccatcagcg cagctattca cccgcagtac
     5341 atagccgcgt ccgccgacat cgaagctgaa ggctcggctc tcttcgccct ccgacaactg
     5401 catcaggtcg ctcacactgt cgaacttctc gatgagaaat ttttcaacgc tggtagcggt
     5461 gagttcgggc ttcttcatgg tggcgctagt gtcagaagaa tcgagctttt tgcaaaagcc
     5521 taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggccg aggcggcctc
     5581 ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg
     5641 cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag
     5701 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc
     5761 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     5821 acaccctaac tgacacacat tccacagctg gttctttccg cctcagaagg tacctaacca
     5881 agttcctctt tcagaggtta tttcaggcca tggtgctgcg c
//
