LOCUS       sequence-478497-        6717 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express HaloTag-AMPKa 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 6717)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6717)
  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            "Unpublished"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: HaloTag-AMPKa Fusion Vector
FEATURES             Location/Qualifiers
     source          1..6717
                     /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"
     regulatory      1061..1070
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1067..1957
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1970..1990
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(3796..3814)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3851..3972
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3862..3881)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3916..3935
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4217..4574
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4217..4408
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4240..4260)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4425..4560
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4589..4636
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4668..5462
                     /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(4722..4741)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5332..5351
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5526..5574
                     /note="synthetic polyadenylation signal"
     rep_origin      5731..6276
                     /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     6177..6196
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6393..6410
                     /label=L4440
                     /note="L4440 vector, forward primer"
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 ggctagcaaa gccaccatgg cagaaatcgg
     1081 tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca tgcactacgt
     1141 cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc cgacctcctc
     1201 ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca ttgctccaga
     1261 cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg acgaccacgt
     1321 ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc tggtcattca
     1381 cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc gcgtcaaagg
     1441 tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc cagaatttgc
     1501 ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga tcatcgatca
     1561 gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga ctgaagtcga
     1621 gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac tgtggcgctt
     1681 cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg tcgaagaata
     1741 catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca ccccaggcgt
     1801 tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact gcaaggctgt
     1861 ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga tcggcagcga
     1921 gatcgcgcgc tggctgtcga cgctcgagat ttccggcgag ccaaccactg aggatctgta
     1981 ctttcagagc gataacgcga tcgccatgcg cagactcagt tcctggagaa agatggcgac
     2041 agccgagaag cagaaacacg acgggcgggt gaagatcggc cactacattc tgggtgacac
     2101 gctgggggtc ggcaccttcg gcaaagtgaa ggttggcaaa catgaattga ctgggcataa
     2161 agtagctgtg aagatactca atcgacagaa gattcggagc cttgatgtgg taggaaaaat
     2221 ccgcagagaa attcagaacc tcaagctttt caggcatcct catataatta aactgtacca
     2281 ggtcatcagt acaccatctg atattttcat ggtgatggaa tatgtctcag gaggagagct
     2341 atttgattat atctgtaaga atggaaggct ggatgaaaaa gaaagtcggc gtctgttcca
     2401 acagatcctt tctggtgtgg attattgtca caggcatatg gtggtccata gagatttgaa
     2461 acctgaaaat gtcctgcttg atgcacacat gaatgcaaag atagctgatt ttggtctttc
     2521 aaacatgatg tcagatggtg aatttttaag aacaagttgt ggctcaccca actatgctgc
     2581 accagaagta atttcaggaa gattgtatgc aggcccagag gtagatatat ggagcagtgg
     2641 ggttattctc tatgctttat tatgtggaac ccttccattt gatgatgacc atgtgccaac
     2701 tctttttaag aagatatgtg atgggatctt ctatacccct caatatttaa atccttctgt
     2761 gattagcctt ttgaaacata tgctgcaggt ggatcccatg aagagggcca caatcaaaga
     2821 tatcagggaa catgaatggt tcaaacagga ccttccaaaa tatctctttc ctgaggatcc
     2881 atcatatagt tcaaccatga ttgatgatga agccttaaaa gaagtatgtg aaaagtttga
     2941 gtgctcagaa gaggaagttc tcagctgtct ttacaacaga aatcaccagg atcctttggc
     3001 agttgcctac catctcataa tagataacag gagaataatg aatgaagcca aagatttcta
     3061 tttggcgaca agcccacctg attcttttct tgatgatcat cacctgactc ggccccatcc
     3121 tgaaagagta ccattcttgg ttgctgaaac accaagggca cgccataccc ttgatgaatt
     3181 aaatccacag aaatccaaac accaaggtgt aaggaaagca aaatggcatt taggaattag
     3241 aagtcaaagt cgaccaaatg atattatggc agaagtatgt agagcaatca aacaattgga
     3301 ttatgaatgg aaggttgtaa acccatatta tttgcgtgta cgaaggaaga atcctgtgac
     3361 aagcacttac tccaaaatga gtctacagtt ataccaagtg gatagtagaa cttatctact
     3421 ggatttccgt agtattgatg atgaaattac agaagccaaa tcagggactg ctactccaca
     3481 gagatcggga tcagttagca actatcgatc ttgccaaagg agtgattcag atgctgaggc
     3541 tcaaggaaaa tcctcagaag tttctcttac ctcatctgtg acctcacttg actcttctcc
     3601 tgttgaccta actccaagac ctggaagtca cacaatagaa ttttttgaga tgtgtgcaaa
     3661 tctaattaaa attcttgcac aagtttaaac gaattcgggc tcggtacccg gggatcctct
     3721 agagtcgacc tgcaggcatg caagctgatc cggctgctaa caaagcccga aaggaagctg
     3781 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcg gccgcttcga
     3841 gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     3901 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     3961 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg
     4021 tgggaggttt ttttaagcaa gtaaaacctc tacaaatgtg gtaaaatcga attttaacaa
     4081 aatattaacg cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4141 tcacaccgca tacgcggatc tgcgcagcac catggcctga aataacctct gaaagaggaa
     4201 cttggttagg taccttctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     4261 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4321 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4381 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4441 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4501 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4561 taggcttttg caaaaagctt aattaactgt tgacaattaa tcatcggcat agtatatcgg
     4621 catagtataa tacgacaagg tgaggaacta aacccaggag gcagatcatg attgaacaag
     4681 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     4741 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     4801 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     4861 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4921 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4981 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5041 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5101 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     5161 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     5221 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     5281 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     5341 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     5401 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     5461 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgatg
     5521 gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga
     5581 tagcgataag gatccgctca tgcgatgata agctgtcaaa catgagaatt acaacttata
     5641 tcgtatgggg ctgacttcag gtgctacatt tgaagagata aattgcactg aaatctagaa
     5701 atattttatc tgattaataa gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc
     5761 ttgctctgaa aacgaaaaaa ccgccttgca gggcggtttt tcgaaggttc tctgagctac
     5821 caactctttg aaccgaggta actggcttgg aggagcgcag tcaccaaaac ttgtcctttc
     5881 agtttagcct taaccggcgc atgacttcaa gactaactcc tctaaatcaa ttaccagtgg
     5941 ctgctgccag tggtgctttt gcatgtcttt ccgggttgga ctcaagacga tagttaccgg
     6001 ataaggcgca gcggtcggac tgaacggggg gttcgtgcat acagtccagc ttggagcgaa
     6061 ctgcctaccc ggaactgagt gtcaggcgtg gaatgagaca aacgcggcca taacagcgga
     6121 atgacaccgg taaaccgaaa ggcaggaaca ggagagcgca cgagggagcc gccaggggga
     6181 aacgcctggt atctttatag tcctgtcggg tttcgccacc actgatttga gcgtcagatt
     6241 tcgtgatgct tgtcaggggg gcggagccta tggaaaaacg gctttgccgc ggccctctca
     6301 cttccctgtt aagtatcttc ctggcatctt ccaggaaatc tccgccccgt tcgtaagcca
     6361 tttccgctcg ccgcagtcga acgaccgagc gtagcgagtc agtgagcgag gaagcggaat
     6421 atatcctgta tcacatattc tgctgacgca ccggtgcagc cttttttctc ctgccacatg
     6481 aagcacttca ctgacaccct catcagtgcc aacatagtaa gccagtatac actccgctag
     6541 cgctgatgtc cggcggtgct tttgccgtta cgcaccaccc cgtcatgaga ttacgcgttt
     6601 tagcggtgcg tacaattaag ggattatggt aaatccactt actgtctgcc ctcgtagcca
     6661 tcgagataaa ccgcagtact ccggccacga tgcgtccggc gtagaggatc gagatct
//
