LOCUS       sequence-475938-        7122 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpression of Halo-NLS-tDeg in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7122)
  AUTHORS   Yang LZ, Min YH, Liu YX, Gao BQ, Liu XQ, Huang Y, Wang H, Yang L, 
            Liu ZJ, Chen LL
  TITLE     CRISPR-array-mediated imaging of non-repetitive and multiplex 
            genomic loci in living cells.
  JOURNAL   Nat Methods. 2024 Sep;21(9):1646-1657. doi: 
            10.1038/s41592-024-02333-3. Epub 2024 Jul 4.
  PUBMED    38965442
REFERENCE   2  (bases 1 to 7122)
  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"; doi: 
            "10.1038/s41592-024-02333-3"; journalName: "Nat Methods"; date: 
            "2024-09"; volume: "21"; issue: "9"; pages: "1646-1657"
COMMENT     Sequence Label: pHAGE-EFS-Halo-NLS-tDeg
FEATURES             Location/Qualifiers
     source          1..7122
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2265..2476
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2530..3414
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             3433..3453
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3512..4100
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3565..3585)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3983..3994)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4175..4408
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(4527..4545)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4613..4717
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4718..4786,4787..5578)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4936..4955)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5749..6337
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6238..6257
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6491..6508
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6748..6769
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6784..6789,6790..6807,6808..6814)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6822..6838
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     6827..6849
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6846..6862
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6846..6862
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6912..6931
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttgacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagcgat
     2221 gctagctgat gcgggccctt cgagtggctc cggtgcccgt cagtgggcag agcgcacatc
     2281 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     2341 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     2401 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     2461 tgccgccaga acacaggtgt cgtgacgcgg ttccggttaa ggtgaacgcg tgactagtgg
     2521 atgggatccg aaatcggtac tggctttcca ttcgaccccc attatgtgga agtcctgggc
     2581 gagcgcatgc actacgtcga tgttggtccg cgcgatggca cccctgtgct gttcctgcac
     2641 ggtaacccga cctcctccta cgtgtggcgc aacatcatcc cgcatgttgc accgacccat
     2701 cgctgcattg ctccagacct gatcggtatg ggcaaatccg acaaaccaga cctgggttat
     2761 ttcttcgacg accacgtccg cttcatggat gccttcatcg aagccctggg tctggaagag
     2821 gtcgtcctgg tcattcacga ctggggctcc gctctgggtt tccactgggc caagcgcaat
     2881 ccagagcgcg tcaaaggtat tgcatttatg gagttcatcc gccctatccc gacctgggac
     2941 gaatggccag aatttgcccg cgagaccttc caggccttcc gcaccaccga cgtcggccgc
     3001 aagctgatca tcgatcagaa cgtttttatc gagggtacgc tgccgatggg tgtcgtccgc
     3061 ccgctgactg aagtcgagat ggaccattac cgcgagccgt tcctgaatcc tgttgaccgc
     3121 gagccactgt ggcgcttccc aaacgagctg ccaatcgccg gtgagccagc gaacatcgtc
     3181 gcgctggtcg aagaatacat ggactggctg caccagtccc ctgtcccgaa gctgctgttc
     3241 tggggcaccc caggcgttct gatcccaccg gccgaagccg ctcgcctggc caaaagcctg
     3301 cctaactgca aggctgtgga catcggcccg ggtctgaatc tgctgcaaga agacaacccg
     3361 gacctgatcg gcagcgagat cgcgcgctgg ctgtctactc tggagatttc cggcgagggt
     3421 tcgtcgctcg agccaaagaa aaagcggaaa gtgagcggac ctagaccaag aggaaccaga
     3481 ggtaagggaa gaaggattcg gcgcaggggg taatcaacct ctggattaca aaatttgtga
     3541 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     3601 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     3661 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     3721 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     3781 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     3841 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     3901 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     3961 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     4021 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     4081 cctttgggcc gcctccccgc ctgagatcct ttaagaccaa tgacttacaa ggcagctgta
     4141 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     4201 agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     4261 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     4321 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     4381 ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat
     4441 ttataacttg caaagaaatg aatatcagag agtgagaggc ccgggttaat taaggaaagg
     4501 gctagatcat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4561 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4621 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4681 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4741 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4801 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4861 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4921 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
     4981 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     5041 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5101 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     5161 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     5221 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     5281 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5341 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5401 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5461 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5521 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5581 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5641 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5701 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5761 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5821 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5881 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5941 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6001 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     6061 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6121 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     6181 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6241 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6301 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6361 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     6421 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     6481 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     6541 tctccccgcg cgttggccga ttcattaatg cagcaagctc atggctgact aatttttttt
     6601 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     6661 ttttttggag gcctaggctt ttgcaaaaag ctccccgtgg cacgacaggt ttcccgactg
     6721 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     6781 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     6841 tcacacagga aacagctatg acatgattac gaatttcaca aataaagcat ttttttcact
     6901 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcaactg
     6961 gataactcaa gctaaccaaa atcatcccaa acttcccacc ccatacccta ttaccactgc
     7021 caattacctg tggtttcatt tactctaaac ctgtgattcc tctgaattat tttcatttta
     7081 aagaaattgt atttgttaaa tatgtactac aaacttagta gt
//
