LOCUS       sequence-361956-        7630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses H2B-GFP in human pluripotent stem cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7630)
  AUTHORS   Price CJ, Barbaric I
  TITLE     Assessing Cell Competition in Human Pluripotent Stem Cell (hPSC) 
            Cultures.
  JOURNAL   Curr Protoc. 2022 May;2(5):e435. doi: 10.1002/cpz1.435.
  PUBMED    35621694
REFERENCE   2  (bases 1 to 7630)
  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: "Curr 
            Protoc."; date: "2022-05"; volume: "2(5):e435. doi"; pages: " 
            10.1002/cpz1.435"
COMMENT     Sequence Label: pCAG-H2B-GFP
FEATURES             Location/Qualifiers
     source          1..7630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    578..712
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        726..1105
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1383..2399
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2323..2345
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2407..2426
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2548..2925
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      2938..2947
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2944..2946,2947..2949,2950..3663)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2944..2946,2947..2949,2950..3660)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2989..3010)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3250..3269)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3597..3618
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(3705..4279,4280..4282,4283..4291)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3872..3889)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4099..4118
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4292..4891
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4292..4311)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4788..4808
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4972..4989)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4978..5202
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5249..5265)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5249..5265)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5262..5284)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5273..5289
                     /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(5297..5303,5304..5321,5322..5327))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5342..5363
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5422..5617
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5468..5603
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5623..5757
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5825..5842)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5996..6584)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6076..6095)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6755..7546,7547..7615))
                     /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     7378..7397
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atgaccgctt tggaaaaaca aagactgtat ttcctggaaa ttaatgttta ttcaataaac
       61 tgtgtattca gctatattca catagtggtg aggctgaaat gaggcgggaa gaggcggttg
      121 gggcttaatt atatcaattt gggtggcccc acagcgcctc caaggcgcca gtcctgtttt
      181 gacaagttgc ctctggaagc ctctcttctt tttctccaga gtaagcggag gccaggggcc
      241 cccggcctct gcttaatact aaaaaaaaca gctgttgtca tagtaatgat tgggtggaaa
      301 cattccaggc ctgggtggag aggctttttg cttcctcttg caaaaccaca ctgacattcc
      361 aggcctgggt ggagaggctt tttgcttcct cttgcaaaac cacactgccc tctggagggc
      421 agttgcctag caactaatta aaagaggatg tcgcacggcc agctgcggtc agttagtcac
      481 ttcctgctta actgacttga cattttctat tttaagagtc gggaggaaaa ttactgtgtt
      541 ggaggccctc cgccatcttc tgaagctgaa tcgaattaac ttgtttattg cagcttataa
      601 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
      661 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tctgatatca
      721 tcgtcgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
      781 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      841 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      901 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      961 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     1021 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     1081 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
     1141 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
     1201 agcgatgggg gcgggggggg ggggggsgcg cgccrggggg gggggggggg ggsgrggggs
     1261 ggggsggggs grggsggrga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     1321 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     1381 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     1441 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     1501 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
     1561 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
     1621 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
     1681 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1741 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1801 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1861 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1921 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     1981 cggggccgcc tcgggccggg grgggctcgg gggaggggcg cggcggcccc cggagcgccg
     2041 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     2101 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     2161 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     2221 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     2281 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     2341 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     2401 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttcctcgaga
     2461 attcatcgat aagcttcccg ggctagcgct accggactca gatctcgagc tcaagcttcg
     2521 aattctgcag tcgacggtac cgccaccatg ccagagccag cgaagtctgc tcccgccccg
     2581 aaaaagggct ccaagaaggc ggtgactaag gcgcagaaga aaggcggcaa gaagcgcaag
     2641 cgcagccgca aggagagcta ttccatctat gtgtacaagg ttctgaagca ggtccaccct
     2701 gacaccggca tttcgtccaa ggccatgggc atcatgaatt cgtttgtgaa cgacattttc
     2761 gagcgcatcg caggtgaggc ttcccgcctg gcgcattaca acaagcgctc gaccatcacc
     2821 tccagggaga tccagacggc cgtgcgcctg ctgctgcctg gggagttggc caagcacgcc
     2881 gtgtccgagg gtactaaggc catcaccaag tacaccagcg ctaaggatcc accggtcgcc
     2941 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     3001 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     3061 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     3121 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     3181 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     3241 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     3301 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     3361 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     3421 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     3481 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     3541 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     3601 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3661 taaagcggcc gctcgataag cttgatatcg aattccgccc cccccccctc tccctccccc
     3721 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     3781 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     3841 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     3901 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     3961 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     4021 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     4081 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     4141 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     4201 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     4261 tgaaaaacac gatgataata tggccacaac catgaccgag tacaagccca cggtgcgcct
     4321 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     4381 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     4441 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     4501 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     4561 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     4621 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     4681 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     4741 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     4801 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     4861 ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac
     4921 cgaaaggagc gcacgacccc atgcatcgat gatctagagc tcgctgatca gcctcgactg
     4981 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     5041 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     5101 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     5161 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     5221 cctgcagccc aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5281 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5341 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5401 acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca accatagtcc cgcccctaac
     5461 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     5521 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     5581 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct
     5641 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5701 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatccgc
     5761 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5821 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5881 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5941 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6001 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6061 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6121 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6181 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6241 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6301 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6361 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6421 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6481 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6541 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6601 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6661 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6721 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6781 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6841 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6901 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6961 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7021 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7081 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7141 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7201 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7261 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7321 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7381 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7441 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7501 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7561 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7621 tcctttttca
//
