LOCUS       sequence-115413-        6135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generate OCT4 mOrange reporter line without selection cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6135)
  AUTHORS   Zhu Z, Verma N, Gonzalez F, Shi ZD, Huangfu D
  TITLE     A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human 
            Embryonic Stem Cells.
  JOURNAL   Stem Cell Reports. 2015 May 28. pii: S2213-6711(15)00128-9. doi: 
            10.1016/j.stemcr.2015.04.016.
  PUBMED    26028531
REFERENCE   2  (bases 1 to 6135)
  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: "Stem Cell 
            Reports. 2015 May 28. pii: S2213-6711(15)00128-9. doi: 
            10.1016/j.stemcr.2015.04.016."
COMMENT     Sequence Label: OCT4-2A-mOrange
FEATURES             Location/Qualifiers
     source          1..6135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1014..1070
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1071..1781
                     /codon_start=1
                     /product="orange monomeric derivative of DsRed fluorescent 
                     protein (Shanet et al., 2004)"
                     /label=mOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKMRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVGIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     1484..1503
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2568..2587)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2569..2587)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2594..2611)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2594..2610)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2603..2625)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2751..3179
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2838..2857)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3048..3069
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             3523..4317
                     /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(3577..3596)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4187..4206
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4335..4403,4404..5195)
                     /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(4553..4572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5366..5954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5855..5874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6108..6125
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gtaccgagct cggatccact agtaacggcc gccagtgtgc tggaattcgc ccttttcctg
      301 caggtcccac ctgcacagat atgcaaagca gaaaccctcg tgcaggcccg aaagagaaag
      361 cgaaccagta tcgagaaccg agtgagaggc aacctggaga atttgttcct gcagtgcccg
      421 aaacccacac tgcagcagat cagccacatc gcccagcagc ttgggctcga gaaggatgtg
      481 agtgccatgt ctctctgcgg gctccatctc tttcccctgt caccacctcg ctttccctag
      541 ctctggctcc tccaactgct ctagggctgt tggctttgga cagaatgtcc aagcagtcag
      601 gcctgtctca gctcattctc taatgtcctc ctctaactgc tctagggctg ttggctttgg
      661 atagaatgtc caagcagagt caggcccgtc tcagctcatt gtctaatgtc attctccttt
      721 ctgtcattca cttgcaggtg gtccgagtgt ggttctgtaa ccggcgccag aagggcaagc
      781 gatcaagcag cgactatgca caacgagagg attttgaggc tgctgggtct cctttctcag
      841 ggggaccagt gtcctttcct ctggccccag ggccccattt tggtacccca ggctatggga
      901 gccctcactt cactgcactg tactcctcgg tccctttccc tgagggggaa gcctttcccc
      961 ctgtctccgt caccactctg ggctctccca tgcattcaaa cgctagcacc ggtgccacga
     1021 acttctctct gttaaagcaa gcaggagacg tggaagaaaa ccccggtcct atggtgagca
     1081 agggcgagga gaataacatg gccatcatca aggagttcat gcgcttcaag gtgcgcatgg
     1141 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1201 agggctttca gaccgctaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     1261 acatcctgtc ccctcagttc acctacggct ccaaggccta cgtgaagcac cccgccgaca
     1321 tccccgacta cttcaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1381 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     1441 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     1501 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     1561 gcgagatcaa gatgaggctg aagctgaagg acggcggcca ctacacctcc gaggtcaaga
     1621 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacatcgtc ggcatcaagt
     1681 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     1741 gccgccactc caccggcggc atggacgagc tgtacaagta aggcgcgccg gtgcctgccc
     1801 ttctaggaat gggggacagg gggaggggag gagctaggga aagaaaacct ggagtttgtg
     1861 ccagggtttt tgggattaag ttcttcattc actaaggaag gaattgggaa cacaaagggt
     1921 gggggcaggg gagtttgggg caactggttg gagggaaggt gaagttcaat gatgctcttg
     1981 attttaatcc cacatcatgt atcacttttt tcttaaataa agaagcctgg gacacagtag
     2041 atagacacac ttatcttggt ttgtccttca gttactgagg tagggatggg aatatccaat
     2101 gctcataccc aagtgaccct gaaactaagg tgccatttac actccttaag gtcacacaac
     2161 atcagaggga gagctgggat tgcagccaag tttatttgta cagggccctg tgataggcta
     2221 gttcccaaaa gcctgtgatg caagaacttt tgcccataga ctcagtcacc atgtagctgt
     2281 tacctgttca gagctggctt tttgctttcc caccctactc tggaattctt aaatggcttt
     2341 atacttagaa atcatcttat ttctgttgaa cctagatcac cccaaccaga aacttctatt
     2401 aatactttgt gctttcttga taccagggtc tatttggttt ccacttaagg tttttgcata
     2461 ctctgcccat aagtgactca ttagttacgg ccggccaaaa gggcgaattc tgcagatatc
     2521 catcacactg gcggccgctc gagcatgcat ctagagggcc caattcgccc tatagtgagt
     2581 cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     2641 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     2701 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg
     2761 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2821 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2881 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2941 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3001 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3061 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3121 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3181 taacaaaatt cagggcgcaa gggctgctaa aggaagcgga acacgtagaa agccagtccg
     3241 cagaaacggt gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac
     3301 gcaagcgcaa agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg
     3361 gcggttttat ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt
     3421 gggaagccct gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg
     3481 ggatcaagat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     3541 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     3601 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     3661 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg
     3721 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     3781 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatcccac
     3841 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     3901 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     3961 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     4021 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg
     4081 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     4141 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     4201 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     4261 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgaatt
     4321 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4441 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4501 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4621 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4741 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4861 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4921 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5041 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5161 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5221 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5281 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5341 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5401 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5461 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5521 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5581 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5641 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5701 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5761 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5821 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5881 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5941 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6001 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6061 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6121 gcgaggaagc ggaag
//
