LOCUS       sequence-356298-        7806 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses human FOXA1 and miRFP670 via T2A linker under control of a
            CMV promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7806)
  AUTHORS   Abatti LE, Lado-Fernandez P, Huynh L, Collado M, Hoffman MM, 
            Mitchell JA
  TITLE     Epigenetic reprogramming of a distal developmental enhancer cluster 
            drives SOX2 overexpression in breast and lung adenocarcinoma.
  JOURNAL   Nucleic Acids Res. 2023 Sep 22:gkad734. doi: 10.1093/nar/gkad734.
  PUBMED    37738673
REFERENCE   2  (bases 1 to 7806)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2023 Sep 22:gkad734. doi: 10.1093/nar/gkad734."
COMMENT     Sequence Label: CMV-FOXA1-T2A-miRFP670
FEATURES             Location/Qualifiers
     source          1..7806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        495..698
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     649..669
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     695..719
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2216..2269
                     /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             2276..3220
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="VAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDHR
                     VIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTEY
                     CGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTGY
                     DRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVTY
                     QPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLVV
                     CHHYLPRFIRFELRAICKRLAERIATRITALES"
     primer_bind     complement(3257..3276)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3301..3349
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3551..3979
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3638..3657)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3848..3869
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3988..4008)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3993..4322
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4173..4308
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4235..4254
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4389..5183
                     /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(4443..4462)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5053..5072
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5359..5480
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5396..5415)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5450..5469
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5529..5545)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5529..5545)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5542..5564)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5553..5569
                     /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(5577..5583,5584..5601,5602..5607))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5622..5643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5760..5777)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5931..6519)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6011..6030)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6690..7481,7482..7550))
                     /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     7313..7332
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7551..7655)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7730..7749)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
       61 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      121 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      181 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      241 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      301 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      361 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      421 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      481 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      541 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      601 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      661 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg
      721 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      781 tccggactct agaatgttag gaactgtgaa gatggaaggg catgaaacca gcgactggaa
      841 cagctactac gcagacacgc aggaggccta ctcctccgtc ccggtcagca acatgaactc
      901 aggcctgggc tccatgaact ccatgaacac ctacatgacc atgaacacca tgactacgag
      961 cggcaacatg accccggcgt ccttcaacat gtcctatgcc aacccgggcc taggggccgg
     1021 cctgagtccc ggcgcagtag ccggcatgcc ggggggctcg gcgggcgcca tgaacagcat
     1081 gactgcggcc ggcgtgacgg ccatgggtac ggcgctgagc ccgagcggca tgggcgccat
     1141 gggtgcgcag caggcggcct ccatgaatgg cctgggcccc tacgcggccg ccatgaaccc
     1201 gtgcatgagc cccatggcgt acgcgccgtc caacctgggc cgcagccgcg cgggcggcgg
     1261 cggcgacgcc aagacgttca agcgcagcta cccgcacgcc aagccgccct actcgtacat
     1321 ctcgctcatc accatggcca tccagcaggc gcccagcaag atgctcacgc tgagcgagat
     1381 ctaccagtgg atcatggacc tcttccccta ttaccggcag aaccagcagc gctggcagaa
     1441 ctccatccgc cactcgctgt ccttcaatga ctgcttcgtc aaggtggcac gctccccgga
     1501 caagccgggc aagggctcct actggacgct gcacccggac tccggcaaca tgttcgagaa
     1561 cggctgctac ttgcgccgcc agaagcgctt caagtgcgag aagcagccgg gggccggcgg
     1621 cgggggcggg agcggaagcg ggggcagcgg cgccaagggc ggccctgaga gccgcaagga
     1681 cccctctggc gcctctaacc ccagcgccga ctcgcccctc catcggggtg tgcacgggaa
     1741 gaccggccag ctagagggcg cgccggcccc cgggcccgcc gccagccccc agactctgga
     1801 ccacagtggg gcgacggcga cagggggcgc ctcggagttg aagactccag cctcctcaac
     1861 tgcgcccccc ataagctccg ggcccggggc gctggcctct gtgcccgcct ctcacccggc
     1921 acacggcttg gcaccccacg agtcccagct gcacctgaaa ggggaccccc actactcctt
     1981 caaccacccg ttctccatca acaacctcat gtcctcctcg gagcagcagc ataagctgga
     2041 cttcaaggca tacgaacagg cactgcaata ctcgccttac ggctctacgt tgcccgccag
     2101 cctgcctcta ggcagcgcct cggtgaccac caggagcccc atcgagccct cagccctgga
     2161 gccggcgtac taccaaggtg tgtattccag acccgtccta aacacttccg ctagcgaggg
     2221 cagaggaagt cttctaacat gcggtgacgt ggaggagaat cccggccctg gtaccgtagc
     2281 aggtcatgcc tctggcagcc ccgcattcgg gaccgcctct cattcgaatt gcgaacatga
     2341 agagatccac ctcgccggct cgatccagcc gcatggcgcg cttctggtcg tcagcgaaca
     2401 tgatcatcgc gtcatccagg ccagcgccaa cgccgcggaa tttctgaatc tcggaagcgt
     2461 actcggcgtt ccgctcgccg agatcgacgg cgatctgttg atcaagatcc tgccgcatct
     2521 cgatcccacc gccgaaggca tgccggtcgc ggtgcgctgc cggatcggca atccctctac
     2581 ggagtactgc ggtctgatgc atcggcctcc ggaaggcggg ctgatcatcg aactcgaacg
     2641 tgccggcccg tcgatcgatc tgtcaggcac gctggcgccg gcgctggagc ggatccgcac
     2701 ggcgggttca ctgcgcgcgc tgtgcgatga caccgtgctg ctgtttcagc agtgcaccgg
     2761 ctacgaccgg gtgatggtgt atcgtttcga tgagcaaggc cacggcctgg tattctccga
     2821 gtgccatgtg cctgggctcg aatcctattt cggcaaccgc tatccgtcgt cgactgtccc
     2881 gcagatggcg cggcagctgt acgtgcggca gcgcgtccgc gtgctggtcg acgtcaccta
     2941 tcagccggtg ccgctggagc cgcggctgtc gccgctgacc gggcgcgatc tcgacatgtc
     3001 gggctgcttc ctgcgctcga tgtcgccgtg ccatctgcag ttcctgaagg acatgggcgt
     3061 gcgcgccacc ctggcggtgt cgctggtggt cggcggcaag ctgtggggcc tggttgtctg
     3121 tcaccattat ctgccgcgct tcatccgttt cgagctgcgg gcgatctgca aacggctcgc
     3181 cgaaaggatc gcgacgcgga tcaccgcgct tgagagctaa accggttagt aatgagttta
     3241 aacgggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     3301 cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttca taaacgcggg
     3361 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     3421 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     3481 gcagccaacg tcggggcggc aggccctgcc atagcagatc tgcgcagctg gggctctagg
     3541 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3601 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3661 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3721 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3781 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3841 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3901 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3961 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     4021 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     4081 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4141 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4201 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4261 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4321 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     4381 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4441 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4501 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4561 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4621 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4681 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4741 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4801 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4861 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     4921 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4981 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     5041 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     5101 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     5161 gccttcttga cgagttcttc tgagcgggac tctggggttc gcgaaatgac cgaccaagcg
     5221 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     5281 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     5341 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     5401 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     5461 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     5521 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5581 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5641 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5701 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5761 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5821 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5881 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5941 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6001 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6061 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6121 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6181 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6241 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6301 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6361 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6421 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6481 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6541 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6601 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6661 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6721 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6781 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6841 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6901 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6961 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7021 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7081 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7141 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7201 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7261 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7321 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7381 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7441 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7501 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7561 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7621 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7681 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     7741 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     7801 agtgcg
//
