LOCUS       sequence-479219-        6922 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express CISH-HaloTag 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 6922)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6922)
  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            "Unpublished"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-HaloTag Fusion Vector
FEATURES             Location/Qualifiers
     source          1..6922
                     /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..1880
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             1896..2780
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(2868..2886)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    2923..3044
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2934..2953)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2988..3007
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3289..3646
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3289..3480
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3312..3332)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3497..3632
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3697..4491
                     /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(3751..3770)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4361..4380
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4555..4603
                     /note="synthetic polyadenylation signal"
     promoter        4759..4863
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4864..4932,4933..5724)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5082..5101)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(5882..6470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5962..5981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6586..6869
                     /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 ttctctcgag ccaaccactg
     1861 aggatctgta ctttcagagc gataacgatg gatccgaaat cggtactggc tttccattcg
     1921 acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg
     1981 atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca
     2041 tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca
     2101 aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct
     2161 tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc
     2221 tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt
     2281 tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg
     2341 ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg
     2401 gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg
     2461 agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa
     2521 tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc
     2581 agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg
     2641 aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc
     2701 tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt
     2761 ctactctgga gatttccggt taatagaatt ctagagtcga cctgcaggca tgcaagctga
     2821 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     2881 actagcataa ccccttgggg cggccgcttc gagcagacat gataagatac attgatgagt
     2941 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     3001 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     3061 ttcattttat gtttcaggtt cagggggaga tgtgggaggt ttttttaagc aagtaaaacc
     3121 tctacaaatg tggtaaaatc gaattttaac aaaatattaa cgcttacaat ttcctgatgc
     3181 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgcgga tctgcgcagc
     3241 accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa
     3301 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3361 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3421 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3481 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3541 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     3601 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttgattcttc
     3661 tgacacaaca gtctcgaact taaggctaga gccaccatga ttgaacaaga tggattgcac
     3721 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3781 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3841 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3901 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3961 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4021 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4081 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4141 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4201 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4261 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     4321 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     4381 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     4441 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4501 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgatgg ccgcaataaa
     4561 atatctttat tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agcgataagg
     4621 atcctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg
     4681 ggtcagcacc gtttctgcgg actggctttc tacgtaatgg tttcttagac gtcaggtggc
     4741 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4801 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4861 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4921 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4981 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5041 cccgaagaac gttttccaat gatgagcact ttcaaagttc tgctatgtgg cgcggtatta
     5101 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5161 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5221 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaact
     5281 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5341 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5401 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     5461 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5521 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     5581 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     5641 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5701 gcctcactga ttaagcattg gtaattcgaa atgaccgacc aagcgacgcc caaccggtat
     5761 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5821 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5881 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5941 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6001 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6061 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6121 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6181 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6241 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6301 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     6361 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6421 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatttcaa gaagatcctt
     6481 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6541 tcatgagatt atcaaaaagg atcttcacct agatcctttt atagtccgga aatacaggaa
     6601 cgcacgctgg atggcccttc gctgggatgg tgaaaccatg aaaaatggca gcttcagtgg
     6661 attaagtggg ggtaatgtgg cctgtaccct ctggttgcat aggtattcat acggttaaaa
     6721 tttatcaggc gcgattgcgg cagtttttcg ggtggtttgt tgccattttt acctgtctgc
     6781 tgccgtgatc gcgctgaacg cgttttagcg gtgcgtacaa ttaagggatt atggtaaatc
     6841 cacttactgt ctgccctcgt agccatcgag ataaaccgca gtactccggc cacgatgcgt
     6901 ccggcgtaga ggatcgagat ct
//
