LOCUS       sequence-454185-        6565 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2A-3xNLS-2xClover mouse Car2 C-term targeting vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6565)
  AUTHORS   Hansen KL, Adachi AS, Braccioli L, Kadvani S, Boileau RM, Martinovic
            M, Pokorny B, Shah R, Anderson EC, Zhang K, Carel I, Bonitto K, 
            Blelloch R, Fudenberg G, de Wit E, Nora EP
  TITLE     Synergy between regulatory elements can render cohesin dispensable 
            for distal enhancer function.
  JOURNAL   Science. 2025 Nov 27:eadt4221. doi: 10.1126/science.adt4221.
  PUBMED    41308125
REFERENCE   2  (bases 1 to 6565)
  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: "Science. 
            2025 Nov 27:eadt4221. doi: 10.1126/science.adt4221."
COMMENT     Sequence Label: pKH032
FEATURES             Location/Qualifiers
     source          1..6565
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1625..1678
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1712..1732
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1736..1756
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1760..1780
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1796..1798,1799..1801,1802..2512)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             join(2543..2545,2546..2548,2549..3259)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     promoter        complement(4377..4395)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4416..4432)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4416..4432)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4429..4451)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4440..4456
                     /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)."
     promoter        complement(join(4464..4470,4471..4488,4489..4494))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4509..4530
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4647..4664)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4818..5406)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4898..4917)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5577..6368,6369..6437))
                     /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     6200..6219
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6438..6542)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacccttta
      661 ctctagaaaa tggtgtgaaa ctatacatat gaattgcatg ttttggcaag atcccatagt
      721 aagtgcatat ttgcatatcg tggtccacaa acaagatctg gaggtcttgt gtacagctca
      781 gagccacagc tctggttcct cctctgagat gccctttgtt ttgctatgct tcttggctct
      841 gttcaacgta ggtggttgaa aaaacctctt gagcaccaca tccaggcaca cccacctctg
      901 aggcagcctc caccgatttc ctgggaacac agaagccttg ttttgaagat cagagatgct
      961 taacaaatct tcccaccatg ctgacgaggc attgtgctcc ttgttcattt ctaagtcaga
     1021 cttgtgagac tccagcaagg gaaagtgtta gcatgaaaga ccaaaactga ctggagttac
     1081 ctatcgtgtt gcactttgtc ttctctgtga cagaaagtac aactttaata tgtatttaaa
     1141 actttatgtt gctttacatg ccataggaat ttacagattt ttgttcgatt taaaaggaaa
     1201 gcaagaaaag caagctcatg aaaatttcta tttaaagaaa gaccccagta tatatttgta
     1261 tagtatatgt aaatagattt ttcatgtgaa ggctgcactt agctggttgt ttgtttgttt
     1321 gttttaattt ttttctttcc cttttttgtc tgcctcccaa gttctgggat caaaggtgta
     1381 cacccctttc cccccttgcc ccccaatgaa ggctgaactt aaatctaaaa catgttgctt
     1441 taagtatgta atattgtgaa actaatttct ttaattacat tgtgtctttt agatgtctca
     1501 tttccgtacg ctgaacttca atgaggaggg ggatgctgaa gaagcgatgg tggacaactg
     1561 gcgtccagct cagccgctaa agaatagaaa gatcaaagcg tcctttaagc tcgagggcag
     1621 tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccaat
     1681 cgatatggcc acaaccatgg tgcacgtgga tccaaaaaag aagagaaagg tagatccaaa
     1741 aaagaagaga aaggtagatc caaaaaagaa gagaaaggta cacgtgtccc atatgatggt
     1801 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1861 cgtaaacggc cacaagttca gcgtccgcgg cgagggcgag ggcgatgcca ccaacggcaa
     1921 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1981 gaccaccttc ggctacggcg tggcctgctt cagccgctac cccgaccaca tgaagcagca
     2041 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tctctttcaa
     2101 ggacgacggt acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     2161 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     2221 ggagtacaac ttcaacagcc acaacgtcta tatcacggcc gacaagcaga agaacggcat
     2281 caaggctaac ttcaagatcc gccacaacgt tgaggacggc agcgtgcagc tcgccgacca
     2341 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2401 gagccatcag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2461 ggagttcgtg accgccgccg ggattacaca tggcatggac gagctgtaca aggggggctc
     2521 aggcggagga tccggttacg taatggtgag caagggcgag gagctgttca ccggggtggt
     2581 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tccgcggcga
     2641 gggcgagggc gatgccacca acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     2701 gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcgtgg cctgcttcag
     2761 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     2821 cgtccaggag cgcaccatct ctttcaagga cgacggtacc tacaagaccc gcgccgaggt
     2881 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     2941 ggacggcaac atcctggggc acaagctgga gtacaacttc aacagccaca acgtctatat
     3001 cacggccgac aagcagaaga acggcatcaa ggctaacttc aagatccgcc acaacgttga
     3061 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     3121 cgtgctgctg cccgacaacc actacctgag ccatcagtcc gccctgagca aagaccccaa
     3181 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga ttacacatgg
     3241 catggacgag ctgtacaagc ctaggaagct tagccatggc ttcccgccgg aggtggagga
     3301 gcaggatgat ggcacgctgc ccatgtcttg tgcccaggag agcgggatgg accgtcaccc
     3361 tgcagcctgt gcttctgcta ggatcaatgt gtaaaagctt gatatcaaca accctgcagc
     3421 aggggtccga agggcacaag tgtgaccgcc tctctgtagc taagcacagt taggctgggt
     3481 gatttggatc ccgactcgca tctggtattg tagacctttt acctctcatc cgttgtgctt
     3541 actaacaaaa tgtgaaaagc aagacccagg tgtctcatgt ggtggcagca cagtggcagg
     3601 ccagtggtca acttagggca tcttttctct gccacggcag cgcaatgcaa agagcagaca
     3661 tggcctcttg cttctcttca cagccatagg ataatgaata ctcaggcctg tttgttaaaa
     3721 tgctattttt aaaaccatat gaaggtagga taattaatta caagtccaca tcatgagaca
     3781 aactgaagta acttaggcaa aacaggtaaa acagtcatag ttttgtgatt ataaatgaga
     3841 tgaatgttca cccttccaag atcttatatt aaagaaaaaa ttttaaaaag cttatatatt
     3901 tgtagcaaag ttattcttaa atatgaatta tgttataact tagtgacttt tgatttctag
     3961 aggtgtaaat gaggatgtaa aaattgatat agttgtgata cagagtatat ttcccttcag
     4021 ataacatacc acaacacaat ggataatgta ttttagatat attctctaat aaaattgaga
     4081 actctattct gggtgcctga ttgtctgcgc cacagggaca gaaatgggtg aacaaatgcc
     4141 tgcctacagg attcagtcag ttcattattg atggtcagcc attacaacag gcttacaacg
     4201 gaaacaaatc cctgcagcca tgtacccact gtttaccagt ggtactttct gagaactggt
     4261 gtgatcctgt aagagtcctg agaagctcgt ctagaaagag agccaggact gatggggcca
     4321 caaaaggaga acctgcttcc gcggccgcca ccgcggtgga gctccagctt ttgttccctt
     4381 tagtgagggt taatttcgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4441 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4501 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4561 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4621 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4681 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4741 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4801 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4861 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4921 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4981 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5041 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5101 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5161 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5221 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5281 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5341 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5401 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5461 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5521 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5581 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5641 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5701 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5761 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5821 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5881 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5941 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6001 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6061 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6121 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6181 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6241 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6301 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6361 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6421 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6481 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6541 cgcacatttc cccgaaaagt gccac
//
