LOCUS       sequence-482147-        7765 bp    DNA     circular SYN 06-AUG-2025
DEFINITION  Constitutive expression of H2B-iRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7765)
  TITLE     Epithelial tension controls intestinal cell extrusion
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7765)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: p2T-H2B-iRFP-IRES-Blast
FEATURES             Location/Qualifiers
     source          1..7765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(150..941,942..1010))
                     /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     773..792
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1011..1115)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1141..1595)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1278..1299)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1490..1509
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1722..1744
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1736..1753
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1737..1753
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1763..1782
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1763..1781
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        2026..2405
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2682..3698
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3622..3644
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3706..3725
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3763..4140
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     CDS             4153..5088
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAG
                     AFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLH
                     RHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVM
                     LYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSR
                     IVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYE
                     PRAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     misc_feature    5131..5683
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5275..5292)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5502..5521
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             5695..6093
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    6125..6259
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6149..6168)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6203..6222
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6429..6449
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     complement(6715..6735)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6715..6733)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6754..6770)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6754..6770)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6767..6789)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6778..6794
                     /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(6802..6808,6809..6826,6827..6832))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6847..6868
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6985..7002)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7156..7744)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7236..7255)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
       61 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
      121 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
      181 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
      241 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
      301 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
      361 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
      421 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
      481 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
      541 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
      601 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
      661 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
      721 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
      781 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
      841 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
      901 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
      961 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     1021 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     1081 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     1141 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     1201 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     1261 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     1321 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     1381 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     1441 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     1501 cgaaaggagc gggcgctagg gcgcggcggg tgtggtggtc acgctgcgcg taaccaccac
     1561 acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     1621 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     1681 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     1741 aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtaccggcat
     1801 atggttcttg acagaggtgt aaaaagtact caaaaatttt actcaagtga aagtacaagt
     1861 acttagggaa aattttactc aattaaaagt aaaagtatct ggctagaatc ttacttgagt
     1921 aaaagtaaaa aagtactcca ttaaaattgt acttgagtat taaggaagta aaagtaaaag
     1981 caagaaagat cgatctcgaa ggatctggag gccacaagct tgctcgacat tgattattga
     2041 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     2101 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     2161 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     2221 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     2281 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     2341 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     2401 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctcccccc
     2461 ccccaatttt gttttttttt tttttttaat tttgtggggg gggggggggg gggggggggg
     2521 gggggcgcgc gccggggggg gcgggggggg gggaggggcg gggcggggcg aggcggagag
     2581 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
     2641 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
     2701 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
     2761 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
     2821 tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg
     2881 agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga
     2941 gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
     3001 tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg
     3061 gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag
     3121 ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg
     3181 agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc
     3241 cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg
     3301 agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga
     3361 gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc
     3421 aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg
     3481 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     3541 gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc
     3601 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     3661 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     3721 ttgtgctgtc tcatcatttt ggcaaagaat tcctcgagca ccatgccaga gccagcgaag
     3781 tctgctcccg ccccgaaaaa gggctccaag aaggcggtga ctaaggcgca gaagaaagac
     3841 ggcaagaagc gcaagcgcag ccgcaaggag agctattcca tctatgtgta caaggttctg
     3901 aagcaggtcc accctgacac cggcatttcg tccaaggcca tgggcatcat gaattcgttt
     3961 gtgaacgaca ttttcgagcg catcgcaggt gaggcttccc gcctggcgca ttacaacaag
     4021 cgctcgacca tcacctccag ggagatccag acggccgtgc gcctgctgct gcctggggag
     4081 ttggccaagc acgccgtgtc cgagggtact aaggccgtca ccaagtacac cagcgctaag
     4141 gatccaccgg tcatggcgcg taaggtcgat ctcacctcct gcgatcgcga gccgatccac
     4201 atccccggca gcattcagcc gtgcggctgc ctgctagcct gcgacgcgca ggcggtgcgg
     4261 atcacgcgca ttacggaaaa tgccggcgcg ttctttggac gcgaaactcc gcgggtcggt
     4321 gagctactcg ccgattactt cggcgagacc gaagcccatg cgctgcgcaa cgcactggcg
     4381 cagtcctccg atccaaagcg accggcgctg atcttcggtt ggcgcgacgg cctgaccggc
     4441 cgcaccttcg acatctcact gcatcgccat gacggtacat cgatcatcga gttcgagcct
     4501 gcggcggccg aacaggccga caatccgctg cggctgacgc ggcagatcat cgcgcgcacc
     4561 aaagaactga agtcgctcga agagatggcc gcacgggtgc cgcgctatct gcaggcgatg
     4621 ctcggctatc accgcgtgat gttgtaccgc ttcgcggacg acggctccgg gatggtgatc
     4681 ggcgaggcga agcgcagcga cctcgagagc tttctcggtc agcactttcc ggcgtcgctg
     4741 gtcccgcagc aggcgcggct actgtacttg aagaacgcga tccgcgtggt ctcggattcg
     4801 cgcggcatca gcagccggat cgtgcccgag cacgacgcct ccggcgccgc gctcgatctg
     4861 tcgttcgcgc acctgcgcag catctcgccc tgccatctcg aatttctgcg gaacatgggc
     4921 gtcagcgcct cgatgtcgct gtcgatcatc attgacggca cgctatgggg attgatcatc
     4981 tgtcatcatt acgagccgcg tgccgtgccg atggcgcagc gcgtcgcggc cgaaatgttc
     5041 gccgacttct tatcgctgca cttcaccgcc gcccaccacc aacgctaagc ggccgcctaa
     5101 gcccgggaat tccgcccctc tcccccccta acgttactgg ccgaagccgc ttggaataag
     5161 gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga
     5221 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg
     5281 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt
     5341 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca
     5401 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc
     5461 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat
     5521 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc
     5581 ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga
     5641 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggcc
     5701 aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc
     5761 atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc
     5821 ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg
     5881 ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag
     5941 aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct
     6001 gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa
     6061 ttgctgccct ctggttatgt gtgggagggc taagtacaag tagtctagtg gatccaccga
     6121 tgatgatcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     6181 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     6241 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     6301 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     6361 tgatcctcta gatcagatct tcgatggatc cagaccacct cccctgcgag cccgtcgacc
     6421 tctagcacac cctaactgac acacattcca cagaattcaa gtgatctcca aaaaataagt
     6481 actttttgac tgtaaataaa attgtaagga gtaaaaagta cttttttttc taaaaaaatg
     6541 taattaagta aaagtaaaag tattgatttt taattgtact caagtaaagt aaaaatcccc
     6601 aaaaataata cttaagtaca gtaatcaagt aaaattactc aagtacttta cacctctggt
     6661 tcttgacccc ctaccttcag caagcccagc agatccgagc tccagctttt gttcccttta
     6721 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     6781 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     6841 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6901 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6961 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     7021 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     7081 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     7141 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     7201 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     7261 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     7321 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     7381 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     7441 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     7501 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     7561 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     7621 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     7681 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     7741 tcaagaagat cctttgatct tttct
//
