LOCUS       sequence-259164-        7212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mouse Klf2 and mCherry via t2A linker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7212)
  AUTHORS   Dhaliwal NK, Abatti LE, Mitchell JA
  TITLE     KLF4 protein stability regulated by interaction with pluripotency 
            transcription factors overrides transcriptional control.
  JOURNAL   Genes Dev. 2019 Jun 20. pii: gad.324319.119. doi: 
            10.1101/gad.324319.119.
  PUBMED    31221664
REFERENCE   2  (bases 1 to 7212)
  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: "Genes Dev. 
            2019 Jun 20. pii: gad.324319.119. doi: 10.1101/gad.324319.119."
COMMENT     Sequence Label: Klf2-t2A-mCherry
FEATURES             Location/Qualifiers
     source          1..7212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1748..1801
                     /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             1808..2512
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWDASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1950..1968)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2218..2237
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2549..2568)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2593..2641
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2843..3271
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2930..2949)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3140..3161
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3280..3300)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3285..3614
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3465..3600
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3527..3546
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3681..4475
                     /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(3735..3754)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4345..4364
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4651..4772
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4688..4707)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4742..4761
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4821..4837)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4821..4837)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4834..4856)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4845..4861
                     /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(4869..4875,4876..4893,4894..4899))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4914..4935
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5052..5069)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5223..5811)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5303..5322)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5982..6773,6774..6842))
                     /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     6605..6624
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6843..6947)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7022..7041)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg gactctagaa tggcgctcag cgagcctatc ttgccgtcct ttgccacttt
      721 cgccagcccg tgcgagcgcg gcctccagga gcgctggccg cgaaatgaac ccgaggcggg
      781 cggcacggat gaggacctaa acaacgtgtt ggacttcatc ctctccatgg gattggacgg
      841 tctgggcgcc gagaatcctc ccgagccccc gccgcagccc ccgccgcctg ccttctacta
      901 cccggagccg ggtgctccgc cgccctacag catccccgcg gccagcctgg gaacagagct
      961 gctgcgcccc gacctggacc cgcctcaggg gccggctctg cacggccgct tcctccttgc
     1021 gcctcccggg cggctagtga aggccgagcc ccccgaggtg gacggcggcg gctacggctg
     1081 cgctccgggc ctggcccacg gaccgcgcgg gctgaagctc gagggcgccc caggagcgac
     1141 aggtgcatgc atgcggggtc ccgccggccg ccccccgccg cccccggaca cgccgccgct
     1201 cagccccgac ggccccctgc gcatcccggc gtccggtcca cgcaacccgt tcccgccgcc
     1261 cttcggtccc ggccccagct tcggcggccc cggccccgcg ttgcactacg ggcctcccgc
     1321 gcctggcgcc ttcggtcttt tcgaggacgc ggcggcagcg gcggcggcgc tgggcttggc
     1381 tccacctgcc acgcgcggtc tcctcacgcc gccctcgtcc ccgctggagc tgctggaggc
     1441 caagcccaaa cgcggccgcc gctcctggcc ccgcaagcgc gccgccacac atacttgcag
     1501 ctacaccaac tgcggcaaga cctacaccaa gagctcgcac ctaaaggcgc atctgcgtac
     1561 acacacaggt gagaagcctt atcattgcaa ctgggaagga tgcggctgga agttcgcgcg
     1621 ctctgacgag cttacccgcc actaccgaaa gcacacaggt cacagaccct ttcagtgcca
     1681 cttgtgcgac cgggccttct ccaggtccga ccacctggcc ttgcacatga agcgacacat
     1741 ggctagcgag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc
     1801 tggtaccgtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     1861 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     1921 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     1981 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     2041 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     2101 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     2161 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     2221 cgtaatgcag aagaagacca tgggctggga tgcctcctcc gagcggatgt accccgagga
     2281 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     2341 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     2401 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     2461 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agaccggtta
     2521 gtaatgagtt taaacggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     2581 cccgcgctat gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt
     2641 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     2701 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     2761 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc
     2821 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2881 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2941 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3001 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3061 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3121 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3181 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3241 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3301 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3361 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3421 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3481 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3541 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3601 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     3661 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3721 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3781 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3841 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     3901 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3961 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4021 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4081 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4141 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4201 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4261 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4321 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4381 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4441 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgcgaaatg
     4501 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     4561 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4621 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4681 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4741 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4801 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4861 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4921 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4981 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5041 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5101 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5161 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5221 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5281 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5341 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5401 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5461 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5521 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5581 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5641 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5701 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5761 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5821 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5881 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5941 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6001 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6061 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6121 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6181 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6241 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6301 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6361 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6421 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6481 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6541 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6601 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6661 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6721 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6781 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6841 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6901 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6961 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     7021 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     7081 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     7141 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     7201 gatatacgcg tt
//
