LOCUS       sequence-453792-        6620 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes H2B-iRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6620)
  AUTHORS   McNamara HM, Solley SC, Adamson B, Chan MM, Toettcher JE
  TITLE     Recording morphogen signals reveals mechanisms underlying gastruloid
            symmetry breaking.
  JOURNAL   Nat Cell Biol. 2024 Oct 2. doi: 10.1038/s41556-024-01521-9.
  PUBMED    39358450
REFERENCE   2  (bases 1 to 6620)
  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: "Nat Cell 
            Biol. 2024 Oct 2. doi: 10.1038/s41556-024-01521-9."
COMMENT     Sequence Label: H2B-iRFP
FEATURES             Location/Qualifiers
     source          1..6620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             81..458
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             513..1472
                     /codon_start=1
                     /product="naturally monomeric infrared fluorescent protein 
                     derived from a truncated bacteriophytochrome (Yu et al., 
                     2015)"
                     /label=mIFP
                     /translation="MSVPLTTSAFGHAFLANCEREQIHLAGSIQPHGILLAVKEPDNVV
                     IQASINAAEFLNTNSVVGRPLRDLGGDLPLQILPHLNGPLHLAPMTLRCTVGSPPRRVD
                     CTIHRPSNGGLIVELEPATKTTNIAPALDGAFHRITSSSSLMGLCDETATIIREITGYD
                     RVMVVRFDEEGNGEILSERRRADLEAFLGNRYPASTIPQIARRLYEHNRVRLLVDVNYT
                     PVPLQPRISPLNGRDLDMSLSCLRSMSPIHQKYMQDMGVGATLVCSLMVSGRLWGLIAC
                     HHYEPRFVPFHIRAAGEALAETCAIRIATLESFAQSQSK"
     misc_feature    1580..2168
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1633..1653)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2051..2062)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   2411..2473
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2585..2604)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2704..2726
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2764..2782)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2850..2954
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2955..3023,3024..3815)
                     /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(3173..3192)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3986..4574
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4475..4494
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4728..4745
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4862..4883
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4898..4903,4904..4921,4922..4928)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4936..4952
                     /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     4941..4963
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4960..4976
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4960..4976
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5011..5045
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    5604..5622
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5625..5643
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     intron          join(5688..6620,1..6)
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6583..6603
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 tttcaggtgt cgtgagggga tctggagctc tcgagaattc tcacgcgtca agtggagcaa
       61 ggcaggtgga cagtggatcc atgccagagc cagcgaagtc tgctcccgcc ccgaaaaagg
      121 gctccaagaa ggcggtgact aaggcgcaga agaaaggcgg caagaagcgc aagcgcagcc
      181 gcaaggagag ctattccatc tatgtgtaca aggttctgaa gcaggtccac cctgacaccg
      241 gcatttcgtc caaggccatg ggcatcatga attcgtttgt gaacgacatt ttcgagcgca
      301 tcgcaggtga ggcttcccgc ctggcgcatt acaacaagcg ctcgaccatc acctccaggg
      361 agatccagac ggccgtgcgc ctgctgctgc ctggggagtt ggccaagcac gccgtgtccg
      421 agggtactaa ggccatcacc aagtacacca gcgctaagaa catgagcggc ggcaccagta
      481 ccagcagctc aggctccggc ggacatactg ggatgtcggt accgctgact acctcagcat
      541 tcggccacgc gtttctggct aactgtgaac gcgagcagat ccacctggcg ggctccattc
      601 agccgcacgg tatcctgctg gctgtgaaag agccggacaa cgtggtgatc caggcttcta
      661 ttaacgctgc ggagttcctg aacaccaact ctgttgttgg ccgtccgctg cgtgacctgg
      721 gcggcgatct gcctttgcag atcctgccgc acctgaacgg cccgctgcac ctggctccga
      781 tgaccctgcg ttgtaccgtg ggttctccgc cgcgtcgtgt ggactgtacc attcatcgtc
      841 cgtctaacgg cggcctgatc gtagaactgg aaccagcaac caagaccact aacattgcgc
      901 cggctctgga cggtgcgttt catcgtatca cttcttcatc ctccctgatg ggcctgtgtg
      961 acgaaaccgc gactattatc cgtgagatta ctggctacga ccgtgtgatg gtagtacgtt
     1021 tcgatgaaga gggtaatggc gaaattctgt ccgaacgtcg tcgtgcggac ctggaagcgt
     1081 tcctgggtaa ccgctacccg gcgtctacta ttccgcagat cgctcgtcgc ctgtacgaac
     1141 ataaccgtgt tcgcctgctg gtagatgtga actatactcc ggttccgcta cagccgcgca
     1201 tcagcccgct gaacggtcgt gatctggata tgtccctgtc ttgcctgcgc tctatgtccc
     1261 cgatccacca gaaatacatg caggacatgg gcgttggcgc gaccctggtt tgctctctga
     1321 tggtgtctgg tcgtctgtgg ggtctgatcg cttgccacca ctacgaaccg cgcttcgttc
     1381 cgttccacat tcgcgctgct ggcgaagcgc tggcggaaac ttgtgcgatc cgcatcgcga
     1441 cgctggagag ctttgcacag tctcagtcca aatagcctgc aggcggccgc aactcccacc
     1501 tgcaacatgc gtgactgact gaggccgcga ctctagagtc gacctgcagg catgcaagct
     1561 tgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     1621 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     1681 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     1741 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     1801 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     1861 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     1921 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     1981 gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     2041 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     2101 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     2161 ctccccgcat cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca
     2221 tgcatcgata ctagttaaaa gttttgttac tttatagaag aaattttgag tttttgtttt
     2281 tttttaataa ataaataaac ataaataaat tgtttgttga atttattatt agtatgtaag
     2341 tgtaaatata ataaaactta atatctattc aaattaataa ataaacctcg atatacagac
     2401 cgataaaaca catgcgtcaa ttttacgcat gattatcttt aacgtacgtc acaatatgat
     2461 tatctttcta gggttaatct agtatacgcg tatgcggccg cttaattaat ccatggcgcc
     2521 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     2581 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     2641 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     2701 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     2761 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     2821 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2881 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2941 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     3001 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     3061 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     3121 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     3181 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     3241 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     3301 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3361 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3421 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3481 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     3541 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3601 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3661 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3721 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3781 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3841 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3901 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3961 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4021 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4081 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     4141 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4201 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4261 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4321 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4381 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4441 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4501 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4561 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4621 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4681 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4741 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4801 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgat cgcaacgcaa
     4861 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     4921 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     4981 attacgccgg ggcgcgccat tctagattaa ccctagaaag atagtctgcg taaaattgac
     5041 gcatgcattc ttgaaatatt gctctctctt tctaaatagc gcgaatccgt cgctgtgcat
     5101 ttaggacatc tcagtcgccg cttggagctc ccgtgaggcg tgcttgtcaa tgcggtaagt
     5161 gtcactgatt ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttttac
     5221 gtgactttta agatttaact catacgataa ttatattgtt atttcatgtt ctacttacgt
     5281 gataacttat tatatatata ttttcttgtt atagatatca actagaatgc tagcatgggc
     5341 ccagatctcc gaggggaccc gacaggcccg cagggacagc agagatccag tttggatcga
     5401 taagcttgat atcgaattgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
     5461 gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag aaggtggcgc
     5521 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
     5581 gaaccgtata taagtgcagg atctccctat cagtgataga gatctcccta tcagtgatag
     5641 agatcgtcga cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt
     5701 gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt
     5761 ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga
     5821 gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg
     5881 ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg
     5941 ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag
     6001 atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg
     6061 gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc
     6121 ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc
     6181 tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg
     6241 cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc
     6301 ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct
     6361 cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt
     6421 tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt
     6481 ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct
     6541 ccttggaatt tgcccttttt aagtttggat cttggttcat tctcaagcct cagacagtgg
     6601 ttcaaagttt ttttcttcca
//
