LOCUS       sequence-271165-        8002 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CENP-A Halo expression construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8002)
  AUTHORS   Swartz SZ, McKay LS, Su KC, Bury L, Padeganeh A, Maddox PS, Knouse 
            KA, Cheeseman IM
  TITLE     Quiescent Cells Actively Replenish CENP-A Nucleosomes to Maintain 
            Centromere Identity and Proliferative Potential.
  JOURNAL   Dev Cell. 2019 Oct 7;51(1):35-48.e7. doi: 
            10.1016/j.devcel.2019.07.016. Epub 2019 Aug 15.
  PUBMED    31422918
REFERENCE   2  (bases 1 to 8002)
  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: 
            "10.1016/j.devcel.2019.07.016"; journalName: "Dev Cell"; date: 
            "2019-10-7- 7"; volume: "51"; issue: "1"; pages: "35-48.e7"
COMMENT     Sequence Label: pKC158
FEATURES             Location/Qualifiers
     source          1..8002
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..135
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    252..273
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(288..293,294..311,312..318)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    326..342
                     /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     331..353
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     350..366
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     350..366
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        387..405
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             1606..1626
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1639..1683
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1708..2598
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     protein_bind    2646..2679
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2744..3243
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2779..2802)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3176..3195
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3255..3302
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3321..4124
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3384..3403)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3994..4013
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4156..4173)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4162..4386
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4443..4476
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5577..5596)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5578..5596)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5603..5620)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5603..5619)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5612..5634)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5760..6215)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5897..5918)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6109..6128
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6242..6346
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6347..6415,6416..7207)
                     /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(6565..6584)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7378..7966
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7867..7886
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
       61 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
      121 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
      181 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
      241 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
      301 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
      361 tatgaccatg attacgccaa gctcgaaatt aaccctcact aaagggaaca aaagctggta
      421 ccgggcccag tcggcggaga caaggttggc taaaggagat ccgaaagctt cagaagagca
      481 cacacctctt gataaggaag ctgcccttca gccgcctggt aagctccggg agcttcccac
      541 caacccctat gccaccctcc tttttttaaa ttttcccagg tattagggac cctactgtct
      601 cttaactaac tggccaaggg acctcttttg taacctggta ggggattctt ttctgaaaac
      661 atgagccctc caagagcacc ttgtaattct ttatggaatt gatttctaac ctctactacc
      721 tttttaaaac tgtagttttt gactgtataa ataggaactc tctcgtttgt ccaccttaga
      781 tcccttttcc tatcctggga gattgcctct catcacccaa ttactggtga tttccccatc
      841 accagtaaga gatccgccag ttcctaagct cccaggagtc aacacctgct cctaattggg
      901 tcctaagatt aagagccagg aaaaaataat ggaaaggaag attcactgga actcatggag
      961 agcatcattg tcccttgaat ctctcttctg ttactcccta ctcctactcc tccccttccc
     1021 cactccttca caggcaagag aaatatgtgt taaattcact cgtggtgtgg acttcaattg
     1081 gcaagcccag gccctattgg ccctacaaga ggtaagaagg caccaaggca caattgggtg
     1141 agggctgagt gggagaaaga ggcagcctag aattctccag tgctgactgg agtgtcacat
     1201 gctgaatctg acctctggaa aagaacaatg aggtaatctc tgagggtttg gaggctggat
     1261 tctgcgtggt gatagcagaa ttccctggca aagttcagtc tcattctgtg aatagtttcc
     1321 tacaatcctt tgagccctca gagattaagt ttagcaggtt tccaaaaaaa agcagcccgt
     1381 ggcccttcat ctgtgtccgt ctttttctct ttctgtctcc aggcagcaga agcatttcta
     1441 gttcatctct ttgaggacgc ctatctcctc accttacatg caggccgagt tactctcttc
     1501 ccaaaggatg tgcaactggc ccggaggatc cggggccttg aggagggact cggcgtcgac
     1561 tccggagggg atcggtggag ctccaccggt ggcggccgct ctagagagaa tctttatttt
     1621 cagggcgccg ccaaattcaa agaaaccgct gctgctaaat tcgaacgcca gcacatggac
     1681 agcggaggtg gaggtactag tctcgacatg gcagaaatcg gtactggctt tccattcgac
     1741 ccccattatg tggaagtcct gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat
     1801 ggcacccctg tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc
     1861 atcccgcatg ttgcaccgac ccatcgctgc attgctccag acctgatcgg tatgggcaaa
     1921 tccgacaaac cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc
     1981 atcgaagccc tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg
     2041 ggtttccact gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc
     2101 atccgcccta tcccgacctg ggacgaatgg ccagaatttg cccgcgagac cttccaggcc
     2161 ttccgcacca ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt tatcgagggt
     2221 acgctgccga tgggtgtcgt ccgcccgctg actgaagtcg agatggacca ttaccgcgag
     2281 ccgttcctga atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc
     2341 gccggtgagc cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag
     2401 tcccctgtcc cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa
     2461 gccgctcgcc tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg
     2521 aatctgctgc aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctttcg
     2581 acgctcgaga tttccggttg agaattcctg cagcccaatt ccgatcatat tcaataaccc
     2641 ttaatataac ttcgtataat gtatgctata cgaagttatt aggtctgaag aggagtttac
     2701 gtccagccaa gctagcttgg ctgcaggtcg tcgaaattct accgggtagg ggaggcgctt
     2761 ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca
     2821 agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc ggctccgttc
     2881 tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt cccccccgcc
     2941 ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac tagtctcgtg
     3001 cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag cggccaatag
     3061 cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc cgggggcggg
     3121 cttaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc
     3181 tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt
     3241 tcgacctgca gcctgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     3301 acaaggtgag gaactaaacc atgggatcgg ccattgaaca agatggattg cacgcaggtt
     3361 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3421 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3481 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3541 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3601 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3661 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3721 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3781 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3841 tcgccaggct caaggcgcgc atgcccgacg gcgatgatct cgtcgtgacc catggcgatg
     3901 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3961 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4021 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4081 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaggggat caattctcta
     4141 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4201 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4261 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4321 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     4381 ctatggcttc tgaggcggaa agaaccagct ggggctcgac tagagcttgc ggaaccctta
     4441 atataacttc gtataatgta tgctatacga agttattagg tccctcgagg ggatccgctc
     4501 ctgcacccag tgtttctgtc agtctttcct gctcagccag ggggtaagct catcctcttt
     4561 cacaggactg gggctggaat ttctcaagta atttcctttc tccatccata gtcccttgtc
     4621 acctcgcctt ccctttgttc tctagtaaag gttgatctag ttttgactgc ctgggacaat
     4681 gtggtttgtg cccttttgtg caatgttaga taaaagatat ctttctatta aataataacc
     4741 agatgaccct aatttctttt ggttgttatt gttgttgttt tatttttata agacagggtc
     4801 tctgtcatca gtgttgccat cgtaactcac tgcaactcct gccttggcct cccaaagtgt
     4861 tgagattaca agaatcagat gacctcattt tgattaagag aaaactttct aataaacttt
     4921 ctaataaaag agaacacttt ctaataaaaa ctttctcttt tttgattaag agaaagtttt
     4981 cttacttttg aaaatggctt gttagtaagt gtgcttcctt aatttcaggg atgataccgg
     5041 ggactctcca gagccatgac tagatccaat ggattctgcg atgctgtctg gactttgctg
     5101 tctctgaaca gtatgtgtgt gttgctttaa atatttttct tttttttgag aaggagaaga
     5161 ctgcatgact ttcctctgta acagaggtaa tatatgagac aatcaacacc gttccaaagg
     5221 cctgaaaata attttcagat aaagagactc caaggttgac tttagtttgt gagttactca
     5281 tgtgactatt tgaggatttt gaaaacatca gatttgctgt ggtatgggag aaaaggttat
     5341 gtacttatta ttttagctct ttctgtaata tttacatttt ttaccatatg tacatttgta
     5401 cttttatttt acacataagg gaaaaaataa gaccactttg agcagttgcc tggaaggctg
     5461 ggcatttcca tcatatagac ctctgccctt cagagtagcc tcaccattag tggcagcatc
     5521 atgtaactga gtggactgtg cttgtcaacg gatgtccgcg gtggagctcc aattcgccct
     5581 atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     5641 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     5701 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     5761 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     5821 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5881 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5941 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     6001 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     6061 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     6121 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     6181 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     6241 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     6301 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     6361 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     6421 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     6481 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     6541 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     6601 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6661 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6721 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6781 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6841 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6901 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6961 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     7021 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     7081 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     7141 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     7201 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     7261 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     7321 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     7381 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     7441 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     7501 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     7561 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     7621 cagtggcgat aagtcgtgtc ttaccgggtt ggacccaaga cgatagttac cggataaggc
     7681 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7741 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7801 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7861 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7921 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7981 ggccttttta cggttcctgg cc
//
