LOCUS       sequence-374266-        8266 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter for BCAM expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8266)
  AUTHORS   Sasamoto Y, Lee CAA, Wilson BJ, Buerger F, Martin G, Mishra A, 
            Kiritoshi S, Tran J, Gonzalez G, Hildebrandt F, Jo VY, Lian CG, 
            Murphy GF, Ksander BR, Frank MH, Frank NY
  TITLE     Limbal BCAM expression identifies a proliferative progenitor 
            population capable of holoclone formation and corneal 
            differentiation.
  JOURNAL   Cell Rep. 2022 Aug 9;40(6):111166. doi: 
            10.1016/j.celrep.2022.111166.
  PUBMED    35947947
REFERENCE   2  (bases 1 to 8266)
  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: "Cell Rep.";
            date: "2022-08-9"; volume: "40(6):111166. doi"; pages: " 
            10.1016/j.celrep.2022.111166"
COMMENT     Sequence Label: BCAM promoter-pRB-Puro-TurboRFPmyc-T2A-Luciferase
FEATURES             Location/Qualifiers
     source          1..8266
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    15..35
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     CDS             2058..2750
                     /codon_start=1
                     /product="red fluorescent protein from Entacmaea 
                     quadricolor"
                     /label=TurboRFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV
                     VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA
                     TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK
                     LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC
                     DLPSKLGHR"
     CDS             2751..2780
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2790..2843
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2844..4493
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
                     IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
                     NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
                     YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
                     DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
                     QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
                     LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
                     YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
                     QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
                     VDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(2907..2925)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     protein_bind    complement(4494..4518)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    4547..4668
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4558..4577)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4612..4631
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        4766..5145
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5146..5349
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5300..5320
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             5382..5981
                     /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(5382..5401)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5878..5898
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6019..6036)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6025..6249
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6274..6291)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6445..7033)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6525..6544)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7204..7995,7996..8064))
                     /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     7827..7846
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8065..8169)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8237..8255
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgcgccgcgg ccgccaactt tgtatagaaa agttggtgca caggctgggg acagaagagc
       61 tagggtatgg gtgtcggtgc tcacagtgtg tgagcttgac agcacctggg actgtcttgg
      121 agtgccgccg cattccccat gagtgagttt gtattgtata tcctgggatg ctccagtctg
      181 gatggaatct gcacgtgagt ttgtgtgcct tgcaaacaaa ccttccctga gcacctgcac
      241 tgtgtctggt cttgtgctgg gcggggtgcc caggggagga gcccttagct caccacagtg
      301 atcaagacat ccctggccct gccctcacag gctcacagtc cactgaggga gacagaccta
      361 tccccagcca gggaccccca gagtgtgccg ggctcaggtg ggggagccca gagggggaac
      421 ctgacccagc cagggaaggc agaggggact tcctggagga ggggatggta cagttaatcc
      481 taaaaggtct ttcccaatgt aacagttgct ctgtaggatg gatgcagtgg ctcatgcctg
      541 taatcccagc actttggaag gcaggaggat cacttgagcc caagagttgg agaccagcct
      601 gggcaacata gtaataccct gtttctatta ccaaaaaaat acaaaaatta gctggacatg
      661 gtaacacaag cctatagtcc cagctacttg ggaggctgaa gtggaaggat tgtttgagcc
      721 caggcggttg aggtttgaat gagctatgat tgcgcccact gcattcccgg ctgggcaaca
      781 gagcgagacc ctgtcaaaaa caaaacaaac aaacaaaaaa aacgttggtc tcctgtatga
      841 ccaatggata aagtaacctt gaccagtcta ataactccca ggctgacctg taatccaagc
      901 cttctctgta tgtctgagca ggcaaagccc agctccaggg ctacctcaac ccccaggctg
      961 atcagaagct tgggtaacct ctcccagtgt ggccagcgac cccccaccct cccacgttct
     1021 gaccactgtc ctgggcaact agtgacatct agtgggggat tttgtcccag tctgacctgg
     1081 cctgtagggg ccttgggcag tcggacctgt gacccacgct tcctagcagt ggctggactc
     1141 ctcccccttc ccgcaggtgc cctggcctct tcccctgccc tcctcacctg ctctggccag
     1201 tctgactcct cccttcccca caaggttggg agggaatgaa ggagagagcg ggaaggagga
     1261 agggagggtg gcattcctgg gattccctgg atagggggaa gagtgggagg gccttccatg
     1321 ggtgagcaag cagagagggc accccgtacc ccacccccag ccccatcatc gaagcggcct
     1381 aggaggacct ggatttgaag accattgaaa ataagagatt aaaccggttg attccaggct
     1441 actggtcaga cgaggaaagc agcccagagc actgaccaca ggggaaaaca tctccgggca
     1501 tactgggagc agctgctggg ggccactggt caggctgcgg gggtcactgg ttggactggg
     1561 agaggccagt gcaggccact cggacggcac gcttggggcc attagttaca ctgggagagg
     1621 ccatcgcagg ccacaggttg cagtggggtc ctctggttaa aatcgacgat gccagcccag
     1681 gctgctggtc acagtgggga cgagtcccca aggcccctgg tcagaataac gaggagaagg
     1741 tccgcgattg cctggtgaga tcccagctcg ggtcactggc tacgctggtc gatcaggccc
     1801 cccgctgcag gcgctggtca gtaagcccgg acagctctag gcttgggggc ggacgagccc
     1861 cgagcctggc cccgtgcact gggaggtccg ctccgggcag tgggaggtcc tggccccgcg
     1921 cccgggcatc gggggacgag gggggcctgg agggcgggcg gggcggggct gccgggggcg
     1981 gggcctctgg ctcccagccc cgcagcggcc gagctgcagc ccgggctcag tctccgccgc
     2041 cgccgtgaac agccaccatg agcgagctga tcaaggagaa catgcacatg aagctgtaca
     2101 tggagggcac cgtgaacaac caccacttca agtgcacatc cgagggcgaa ggcaagccct
     2161 acgagggcac ccagaccatg aagatcaagg tggtcgaggg cggccctctc cccttcgcct
     2221 tcgacatcct ggctaccagc ttcatgtacg gcagcaaagc cttcatcaac cacacccagg
     2281 gcatccccga cttctttaag cagtccttcc ctgagggctt cacatgggag agaatcacca
     2341 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcttccag aacggctgca
     2401 tcatctacaa cgtcaagatc aacggggtga acttcccatc caacggccct gtgatgcaga
     2461 agaaaacacg cggctgggag gccaacaccg agatgctgta ccccgctgac ggcggcctga
     2521 gaggccacag ccagatggcc ctgaagctcg tgggcggggg ctacctgcac tgctccttca
     2581 agaccacata cagatccaag aaacccgcta agaacctcaa gatgcccggc ttccacttcg
     2641 tggaccacag actggaaaga atcaaggagg ccgacaaaga gacctacgtc gagcagcacg
     2701 agatggctgt ggccaagtac tgcgacctcc ctagcaaact ggggcacaga gaacagaaac
     2761 tgatcagcga ggaggatctg ggctccggag agggccgggg aagcctcttg acctgcggcg
     2821 atgtggaaga gaaccccgga cctgaagacg ccaaaaacat aaagaaaggc ccggcgccat
     2881 tctatccgct ggaagatgga accgctggag agcaactgca taaggctatg aagagatacg
     2941 ccctggttcc tggaacaatt gcttttacag atgcacatat cgaggtggac atcacttacg
     3001 ctgagtactt cgaaatgtcc gttcggttgg cagaagctat gaaacgatat gggctgaata
     3061 caaatcacag aatcgtcgta tgcagtgaaa actctcttca attctttatg ccggtgttgg
     3121 gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga catttataat gaacgtgaat
     3181 tgctcaacag tatgggcatt tcgcagccta ccgtggtgtt cgtttccaaa aaggggttgc
     3241 aaaaaatttt gaacgtgcaa aaaaagctcc caatcatcca aaaaattatt atcatggatt
     3301 ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt cgtcacatct catctacctc
     3361 ccggttttaa tgaatacgat tttgtgccag agtccttcga tagggacaag acaattgcac
     3421 tgatcatgaa ctcctctgga tctactggtc tgcctaaagg tgtcgctctg cctcatagaa
     3481 ctgcctgcgt gagattctcg catgccagag atcctatttt tggcaatcaa atcattccgg
     3541 atactgcgat tttaagtgtt gttccattcc atcacggttt tggaatgttt actacactcg
     3601 gatatttgat atgtggattt cgagtcgtct taatgtatag atttgaagaa gagctgtttc
     3661 tgaggagcct tcaggattac aagattcaaa gtgcgctgct ggtgccaacc ctattctcct
     3721 tcttcgccaa aagcactctg attgacaaat acgatttatc taatttacac gaaattgctt
     3781 ctggtggcgc tcccctctct aaggaagtcg gggaagcggt tgccaagagg ttccatctgc
     3841 caggtatcag gcaaggatat gggctcactg agactacatc agctattctg attacacccg
     3901 agggggatga taaaccgggc gcggtcggta aagttgttcc attttttgaa gcgaaggttg
     3961 tggatctgga taccgggaaa acgctgggcg ttaatcaaag aggcgaactg tgtgtgagag
     4021 gtcctatgat tatgtccggt tatgtaaaca atccggaagc gaccaacgcc ttgattgaca
     4081 aggatggatg gctacattct ggagacatag cttactggga cgaagacgaa cacttcttca
     4141 tcgttgaccg cctgaagtct ctgattaagt acaaaggcta tcaggtggct cccgctgaat
     4201 tggaatccat cttgctccaa caccccaaca tcttcgacgc aggtgtcgca ggtcttcccg
     4261 acgatgacgc cggtgaactt cccgccgccg ttgttgtttt ggagcacgga aagacgatga
     4321 cggaaaaaga gatcgtggat tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg
     4381 gaggagttgt gtttgtggac gaagtaccga aaggtcttac cggaaaactc gacgcaagaa
     4441 aaatcagaga gatcctcata aaggccaaga agggcggaaa gatcgccgtg taaacccagc
     4501 tttcttgtac aaagtggtga tggccggccg cttcgagcag acatgataag atacattgat
     4561 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     4621 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     4681 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     4741 aacctctaca aatgtggtac gcgttgacat tgattattga ctagttatta atagtaatca
     4801 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     4861 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     4921 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     4981 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     5041 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     5101 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     5161 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     5221 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     5281 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     5341 agcagagctc tctggctaac tagagaaccc actgcgccac catgaccgag tacaagccca
     5401 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     5461 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     5521 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     5581 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     5641 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     5701 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     5761 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     5821 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     5881 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     5941 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg actcgagtct agagggcccg
     6001 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     6061 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     6121 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     6181 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     6241 gctctatggg cggccgcggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6301 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6361 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6421 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6481 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6541 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6601 gtccgccttt ctctcttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6661 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6721 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6781 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6841 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6901 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6961 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     7021 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     7081 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     7141 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     7201 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     7261 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7321 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7381 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7441 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7501 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7561 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7621 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7681 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7741 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7801 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7861 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7921 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7981 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     8041 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     8101 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     8161 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     8221 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcgg
//
