LOCUS       sequence-213812-        7696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mouse Trex2 and  BFP reporter in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7696)
  AUTHORS   Yumlu S, Bashir S, Stumm J, Kuhn R
  TITLE     Efficient Gene Editing of Human Induced Pluripotent Stem Cells Using
            CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2019;1961:137-151. doi: 
            10.1007/978-1-4939-9170-9_10.
  PUBMED    30912045
REFERENCE   2  (bases 1 to 7696)
  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: "Methods Mol
            Biol."; date: "2019"; volume: "1961:137-151. doi"; pages: " 
            10.1007/978-1-4939-9170-9_10"
COMMENT     Sequence Label: pCAG-Trex2-bpA-EF1BFP
FEATURES             Location/Qualifiers
     source          1..7696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..18
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2..18
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        38..417
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        419..696
                     /label=chicken beta-actin promoter
     intron          697..1713
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1637..1659
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        1723..1741
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2480..2496)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2480..2496)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     polyA_signal    2516..2740
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2777..2795
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2800..2809
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2812..2832
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          2991..3929
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3886..3906
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3964..3973
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3970..3972,3973..3975,3976..4689)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(4015..4036)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4720..4831
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4949..4966)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4949..4965)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4958..4980)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5106..5561)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5243..5264)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5455..5474
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5588..5692
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5693..5761,5762..6553)
                     /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(5911..5930)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6724..7312
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7213..7232
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7466..7483
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7600..7621
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7636..7641,7642..7659,7660..7666)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7674..7690
                     /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     join(7679..7696,1..5)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 acaggaaaca gctatgacca tgaggcgcgc cggattcgac attgattatt gactagttat
       61 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      121 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      181 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      241 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      301 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      361 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      421 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      481 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      541 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      601 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      661 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      721 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      781 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
      841 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc
      901 cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc
      961 gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     1021 gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg
     1081 ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt
     1141 gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt tgctgagcac
     1201 ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc
     1261 ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc
     1321 tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc
     1381 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc
     1441 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag
     1501 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc
     1561 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg
     1621 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
     1681 aaccatgttc atgccttctt ctttttccta cagatccttt aataatacga ctcactatag
     1741 gggccgccac catgtctgag ccaccccggg ctgagacctt tgtattcctg gacctagaag
     1801 ccactgggct cccaaacatg gatcctgaga ttgcagagat atcccttttt gctgttcacc
     1861 gctcttccct ggagaaccca gaacgggatg attctggttc cttggtgctg ccccgtgttc
     1921 tggacaagct cacactgtgc atgtgcccgg agcgcccctt tactgccaag gccagtgaga
     1981 ttactggttt gagcagcgaa agcctgatgc actgcgggaa ggctggtttc aatggcgctg
     2041 tggtaaggac actgcagggc ttcctaagcc gccaggaggg ccccatctgc cttgtggccc
     2101 acaatggctt cgattatgac ttcccactgc tgtgcacgga gctacaacgt ctgggtgccc
     2161 atctgcccca agacactgtc tgcctggaca cactgcctgc attgcggggc ctggaccgtg
     2221 ctcacagcca cggcaccagg gctcaaggcc gcaaaagcta cagcctggcc agtctcttcc
     2281 accgctactt ccaggctgaa cccagtgctg cccattcagc agaaggtgat gtgcacaccc
     2341 tgcttctgat cttcctgcat cgtgctcctg agctgctcgc ctgggcagat gagcaggccc
     2401 gcagctgggc tcatattgag cccatgtacg tgccacctga tggtccaagc ctcgaagcct
     2461 gaacgcgtaa atgattgcag atccactagt tctagagctc gctgatcagc ctcgactgtg
     2521 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2581 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2641 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2701 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2761 agctggggct cgataataat acgactcact ataggggccg ccaccatggg acctaagaaa
     2821 aagaggaagg tggcggcccc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
     2881 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
     2941 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
     3001 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     3061 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     3121 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     3181 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     3241 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     3301 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     3361 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     3421 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg
     3481 gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     3541 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     3601 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     3661 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     3721 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     3781 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     3841 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     3901 tggttcaaag tttttttctt ccatttcagg tgtcgtgagc gatcgcatcg acagtgttta
     3961 aacgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     4021 gagctggacg gcgacgtaaa cggccacaag ttcagcgtga ggggcgaggg cgagggcgat
     4081 gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     4141 tggcccaccc tcgtgaccac cctgagccac ggcgtgcagt gcttcgcccg ctaccccgac
     4201 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     4261 accatcttct tcaaggacga cggcacctac aagacccgcg ccgaggtgaa gttcgagggc
     4321 gacaccctgg tgaaccgcat cgagctgaag ggcgtcgact tcaaggagga cggcaacatc
     4381 ctggggcaca agctggagta caacttcaac agccacaaca tctatatcat ggccgtcaag
     4441 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acgtggagga cggcagcgtg
     4501 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     4561 gacagccact acctgagcac ccagtccgtg ctgagcaaag accccaacga gaagcgcgat
     4621 cacatggtcc tgctggagtt ccgcaccgcc gccgggatca ctctcggcat ggacgagctg
     4681 tacaagtgag aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     4741 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     4801 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     4861 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg
     4921 cgtttaaaca ttagtcgaca gggccgccac tggccgtcgt tttacaacgt cgtgactggg
     4981 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     5041 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     5101 aatggaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5161 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5221 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5281 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     5341 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     5401 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     5461 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     5521 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     5581 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5641 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5701 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5761 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5821 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5881 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5941 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6001 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6061 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6121 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6181 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6241 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6301 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6361 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6421 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6481 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6541 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6601 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6661 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6721 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6781 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6841 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6901 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6961 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7021 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7081 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7141 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7201 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7261 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7321 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     7381 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     7441 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     7501 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     7561 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     7621 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     7681 cggataacaa tttcac
//
