LOCUS       sequence-454190-        6529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2A-3xNLS-tdTomato mouse Car2 C-term targeting vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6529)
  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 6529)
  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: pKH040
FEATURES             Location/Qualifiers
     source          1..6529
                     /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             1796..3223
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     promoter        complement(4341..4359)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4380..4396)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4380..4396)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4393..4415)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4404..4420
                     /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(4428..4434,4435..4452,4453..4458))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4473..4494
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4611..4628)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4782..5370)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4862..4881)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5541..6332,6333..6401))
                     /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     6164..6183
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6402..6506)
                     /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 gaggaggtca tcaaagagtt catgcgcttc aaggtgcgca tggagggctc
     1861 catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     1921 ccagaccgcc aagctgaagg tgaccaaggg cggccccctg cccttcgcct gggacatcct
     1981 gtccccccag ttcatgtacg gctccaaggc gtacgtgaag caccccgccg acatccccga
     2041 ttacaagaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     2101 cggcggtctg gtgaccgtga cccaggactc ctccctgcag gacggcacgc tgatctacaa
     2161 ggtgaagatg cgcggcacca acttcccccc cgacggcccc gtaatgcaga agaagaccat
     2221 gggctgggag gcctccaccg agcgcctgta cccccgcgac ggcgtgctga agggcgagat
     2281 ccaccaggcc ctgaagctga aggacggcgg ccactacctg gtggagttca agaccatcta
     2341 catggccaag aagcccgtgc aactgcccgg ctactactac gtggacacca agctggacat
     2401 cacctcccac aacgaggact acaccatcgt ggaacagtac gagcgctccg agggccgcca
     2461 ccacctgttc ctggggcatg gcaccggcag caccggcagc ggcagctccg gcaccgcctc
     2521 ctccgaggac aacaacatgg ccgtcatcaa agagttcatg cgcttcaagg tgcgcatgga
     2581 gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     2641 gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga
     2701 catcctgtcc ccccagttca tgtacggctc caaggcgtac gtgaagcacc ccgccgacat
     2761 ccccgattac aagaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     2821 cgaggacggc ggtctggtga ccgtgaccca ggactcctcc ctgcaggacg gcacgctgat
     2881 ctacaaggtg aagatgcgcg gcaccaactt cccccccgac ggccccgtaa tgcagaagaa
     2941 gaccatgggc tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg
     3001 cgagatccac caggccctga agctgaagga cggcggccac tacctggtgg agttcaagac
     3061 catctacatg gccaagaagc ccgtgcaact gcccggctac tactacgtgg acaccaagct
     3121 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc gctccgaggg
     3181 ccgccaccac ctgttcctgt acggcatgga cgagctgtac aagcctagga agcttagcca
     3241 tggcttcccg ccggaggtgg aggagcagga tgatggcacg ctgcccatgt cttgtgccca
     3301 ggagagcggg atggaccgtc accctgcagc ctgtgcttct gctaggatca atgtgtaaaa
     3361 gcttgatatc aacaaccctg cagcaggggt ccgaagggca caagtgtgac cgcctctctg
     3421 tagctaagca cagttaggct gggtgatttg gatcccgact cgcatctggt attgtagacc
     3481 ttttacctct catccgttgt gcttactaac aaaatgtgaa aagcaagacc caggtgtctc
     3541 atgtggtggc agcacagtgg caggccagtg gtcaacttag ggcatctttt ctctgccacg
     3601 gcagcgcaat gcaaagagca gacatggcct cttgcttctc ttcacagcca taggataatg
     3661 aatactcagg cctgtttgtt aaaatgctat ttttaaaacc atatgaaggt aggataatta
     3721 attacaagtc cacatcatga gacaaactga agtaacttag gcaaaacagg taaaacagtc
     3781 atagttttgt gattataaat gagatgaatg ttcacccttc caagatctta tattaaagaa
     3841 aaaattttaa aaagcttata tatttgtagc aaagttattc ttaaatatga attatgttat
     3901 aacttagtga cttttgattt ctagaggtgt aaatgaggat gtaaaaattg atatagttgt
     3961 gatacagagt atatttccct tcagataaca taccacaaca caatggataa tgtattttag
     4021 atatattctc taataaaatt gagaactcta ttctgggtgc ctgattgtct gcgccacagg
     4081 gacagaaatg ggtgaacaaa tgcctgccta caggattcag tcagttcatt attgatggtc
     4141 agccattaca acaggcttac aacggaaaca aatccctgca gccatgtacc cactgtttac
     4201 cagtggtact ttctgagaac tggtgtgatc ctgtaagagt cctgagaagc tcgtctagaa
     4261 agagagccag gactgatggg gccacaaaag gagaacctgc ttccgcggcc gccaccgcgg
     4321 tggagctcca gcttttgttc cctttagtga gggttaattt cgagcttggc gtaatcatgg
     4381 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4441 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4501 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4561 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4621 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4681 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4741 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4801 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4861 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4921 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4981 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5041 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5101 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5161 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5221 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5281 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5341 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5401 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5461 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5521 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5581 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5641 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5701 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5761 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5821 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5881 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5941 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6001 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6061 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6121 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6181 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6241 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6301 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6361 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6421 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6481 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
