LOCUS       sequence-462856-        7572 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lac repressor (LacI) which binds lacO sites; labeled with HaloTag 
            and coupled with Kruppel-associated box (KRAB) repressor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7572)
  TITLE     HP1 binding creates a local barrier against transcription activation
            and persists during chromatin decondensation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7572)
  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            "Unpublished"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: HaloTag-LacI-KRAB
FEATURES             Location/Qualifiers
     source          1..7572
                     /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(2028..2047)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3104..3124
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3176..3370
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             3437..3457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3523..3541)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3573..3694
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3584..3603)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3638..3657
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3939..4296
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3939..4130
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3962..3982)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4147..4282
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4347..5141
                     /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(4401..4420)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5011..5030
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5205..5253
                     /note="synthetic polyadenylation signal"
     promoter        5409..5513
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5514..5582,5583..6374)
                     /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(5732..5751)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(6532..7120)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6612..6631)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    7236..7519
                     /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 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 tcgaaatggt gaaaccagta acgttatacg atgtcgcaga
     2041 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     2101 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     2161 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     2221 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     2281 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     2341 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     2401 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     2461 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     2521 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     2581 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     2641 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     2701 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     2761 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     2821 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     2881 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     2941 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     3001 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     3061 acaggtttcc cgaagcagcc tggggcgtcg actgggtacc cctcctaaga agaagaggaa
     3121 ggttgccccc accgaaccgg tcgccaccga tgctaagtca ctgactgcct ggtcccggac
     3181 actggtgacc ttcaaggatg tgtttgtgga cttcaccagg gaggagtgga agctgctgga
     3241 cactgctcag cagatcctgt acagaaatgt gatgctggag aactataaga acctggtttc
     3301 cttgggttat cagcttacta agccagatgt gatcctccgg ttggagaagg gagaagagcc
     3361 ctggctggtg gagagagaaa ttcaccaaga gacccatcct gattcagaga ctgcatttga
     3421 aatcaaatca tcagttccga aaaagaaacg caaagttgcg gccgcgactc tagatcataa
     3481 tgagcagaca tgagcggccg cttgctgagt tggctgctgc caccgctgag caataactag
     3541 cataacccct tggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac
     3601 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     3661 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     3721 gtttcaggtt cagggggaga tgtgggaggt ttttttaagc aagtaaaacc tctacaaatg
     3781 tggtaaaatc gaattttaac aaaatattaa cgcttacaat ttcctgatgc ggtattttct
     3841 ccttacgcat ctgtgcggta tttcacaccg catacgcgga tctgcgcagc accatggcct
     3901 gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg
     3961 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4021 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     4081 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     4141 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4201 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     4261 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttgattcttc tgacacaaca
     4321 gtctcgaact taaggctaga gccaccatga ttgaacaaga tggattgcac gcaggttctc
     4381 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     4441 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     4501 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     4561 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4621 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4681 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4741 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4801 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4861 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4921 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4981 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     5041 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     5101 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     5161 aatgaccgac caagcgacgc ccaacctgcc atcacgatgg ccgcaataaa atatctttat
     5221 tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agcgataagg atcctctttg
     5281 cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc
     5341 gtttctgcgg actggctttc tacgtaatgg tttcttagac gtcaggtggc acttttcggg
     5401 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5461 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5521 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5581 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5641 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5701 gttttccaat gatgagcact ttcaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5761 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5821 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5881 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaact atcggaggac
     5941 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6001 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6061 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6121 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6181 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6241 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6301 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6361 ttaagcattg gtaattcgaa atgaccgacc aagcgacgcc caaccggtat cagctcactc
     6421 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     6481 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     6541 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6601 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6661 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6721 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6781 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6841 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6901 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6961 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     7021 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     7081 ttgcaagcag cagattacgc gcagaaaaaa aggatttcaa gaagatcctt tgatcttttc
     7141 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     7201 atcaaaaagg atcttcacct agatcctttt atagtccgga aatacaggaa cgcacgctgg
     7261 atggcccttc gctgggatgg tgaaaccatg aaaaatggca gcttcagtgg attaagtggg
     7321 ggtaatgtgg cctgtaccct ctggttgcat aggtattcat acggttaaaa tttatcaggc
     7381 gcgattgcgg cagtttttcg ggtggtttgt tgccattttt acctgtctgc tgccgtgatc
     7441 gcgctgaacg cgttttagcg gtgcgtacaa ttaagggatt atggtaaatc cacttactgt
     7501 ctgccctcgt agccatcgag ataaaccgca gtactccggc cacgatgcgt ccggcgtaga
     7561 ggatcgagat ct
//
