LOCUS       sequence-395519-        7295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector For Twist.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7295)
  AUTHORS   Murawala P
  TITLE     Murawala Lab Plasmids 2023
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 7295)
  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: 
            "unpublished"
COMMENT     Sequence Label: MCS-T2a-GFPnls-ER-Cre-ER
FEATURES             Location/Qualifiers
     source          1..7295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..38
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     30..47
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     31..47
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             96..149
                     /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             join(150..152,153..155,156..866)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(150..152,153..155,156..866)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(456..475)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     803..824
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             891..911
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             915..935
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             939..959
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             978..1031
                     /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             1980..3002
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="NLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLS
                     VCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPR
                     PSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLG
                     IAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERW
                     ISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRY
                     LAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDG
                     D"
     CDS             3015..3950
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             3063..3956
                     /codon_start=1
                     /gene="ESR1"
                     /product="estrogen nuclear receptor alpha ligand-binding 
                     domain"
                     /label=ER-LBD
                     /translation="KRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM
                     GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKL
                     LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF
                     LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNK
                     GMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSL
                     QKYYITGEAEGFPATA"
     primer_bind     complement(4040..4059)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4105..4160
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4159..4178)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     promoter        complement(4490..4508)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4491..4508)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4526..4542)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4526..4542)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4539..4561)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4550..4566
                     /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(4574..4580,4581..4598,4599..4604))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4619..4640
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4757..4774)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4928..5516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5008..5027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5687..6478,6479..6547))
                     /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     6310..6329
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6548..6652)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6730..7185)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6867..6888)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7079..7098
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg
       61 actcactata gacgcgtatg gctcccatgg catgcgaggg cagaggaagt cttctaacat
      121 gcggtgacgt ggaggagaat cccggcccta tggtgagcaa gggcgaggag ctgttcaccg
      181 gggtggtacc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
      241 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      301 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
      361 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
      421 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
      481 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
      541 tcaaggagga cggcaacatc ctggggcaca agctagagta caactacaac agccacaacg
      601 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
      661 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
      721 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
      781 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
      841 ctctcggcat ggacgagctg tacaagtccg gactcagatc tcgagctgat ccaaaaaaga
      901 agagaaaggt agatccaaaa aagaagagaa aggtagatcc aaaaaagaag agaaaggtag
      961 gatccaccgg atctagagag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga
     1021 atcccggccc tatggccggc gatatgagag ccgctaacct gtggccctcc ccactgatga
     1081 tcaagagaag caagaagaac tccctggccc tgagcctgac cgctgatcag atggtgtccg
     1141 ccctgctgga cgccgagccc cctatcctgt acagcgagta cgaccccacc aggcccttca
     1201 gcgaggccag catgatggga ctgctgacaa acctggccga tcgcgaactg gtgcatatga
     1261 tcaattgggc caagcgcgtg cccggattcg tggacctgac actgcacgac caggtgcacc
     1321 tgctggaatg cgcttggctg gaaattctga tgatcggcct cgtgtggcgg agcatggaac
     1381 atcctgtgaa gctgctgttc gcccccaatc tgctgctgga ccgcaaccag ggcaaatgcg
     1441 tggaagggat ggtcgaaatc tttgatatgc tgctcgccac cagcagcagg ttcagaatga
     1501 tgaacctcca gggcgaagag ttcgtctgcc tgaagtccat catcctgctg aacagcggcg
     1561 tgtacacctt cctgtcctct accctgaagt ccctcgagga aaaggatcac atccacaggg
     1621 tgctggataa gatcaccgat accctgatcc atctcatggc taaggccgga ctcacactcc
     1681 agcagcagca tcagagactc gctcagctgc tgctgatcct gagccacatc cgccacatgt
     1741 ccaacaaggg aatggaacac ctctactcta tgaagtgtaa aaacgtcgtc cccctgtacg
     1801 atctgctgct cgaggccgcc gatgcccaca gactgcacgc ccctacaagc agaggcggcg
     1861 ctagcgtgga agagacagac cagtcccacc tggccaccgc cggcagcaca agcagccaca
     1921 gcctgcagaa gtactatatc accggcgagg ccgagggctt ccccgctaca gctgtcgaca
     1981 atttactgac cgtacaccaa aatttgcctg cattaccggt cgatgcaacg agtgatgagg
     2041 ttcgcaagaa cctgatggac atgttcaggg atcgccaggc gttttctgag catacctgga
     2101 aaatgcttct gtccgtttgc cggtcgtggg cggcatggtg caagttgaat aaccggaaat
     2161 ggtttcccgc agaacctgaa gatgttcgcg attatcttct atatcttcag gcgcgcggtc
     2221 tggcagtaaa aactatccag caacatttgg gccagctaaa catgcttcat cgtcggtccg
     2281 ggctgccacg accaagtgac agcaatgctg tttcactggt tatgcggcgg atccgaaaag
     2341 aaaacgttga tgccggtgaa cgtgcaaaac aggctctagc gttcgaacgc actgatttcg
     2401 accaggttcg ttcactcatg gaaaatagcg atcgctgcca ggatatacgt aatctggcat
     2461 ttctggggat tgcttataac accctgttac gtatagccga aattgccagg atcagggtta
     2521 aagatatctc acgtactgac ggtgggagaa tgttaatcca tattggcaga acgaaaacgc
     2581 tggttagcac cgcaggtgta gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc
     2641 gatggatttc cgtctctggt gtagctgatg atccgaataa ctacctgttt tgccgggtca
     2701 gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct atcaactcgc gccctggaag
     2761 ggatttttga agcaactcat cgattgattt acggcgctaa ggatgactcc ggtcagagat
     2821 acctggcctg gtctggacac agtgcccgtg tcggagccgc gcgagatatg gcccgcgctg
     2881 gagtttcaat accggagatc atgcaagctg gtggctggac caatgtaaat attgtcatga
     2941 actatatccg taacctggat agtgaaacag gggcaatggt gcgcctgctg gaagatggcg
     3001 atctcgagcc atctgctgga gacatgagag ctgccaacct ttggccaagc ccgctcatga
     3061 tcaaacgctc taagaagaac agcctggcct tgtccctgac ggccgaccag atggtcagtg
     3121 ccttgttgga tgctgagccc cccatactct attccgagta tgatcctacc agacccttca
     3181 gtgaagcttc gatgatgggc ttactgacca acctggcaga cagggagctg gttcacatga
     3241 tcaactgggc gaagagggtg ccaggctttg tggatttgac cctccatgat caggtccacc
     3301 ttctagaatg tgcctggcta gagatcctga tgattggtct cgtctggcgc tccatggagc
     3361 acccagtgaa gctactgttt gctcctaact tgctcttgga caggaaccag ggaaaatgtg
     3421 tagagggcat ggtggagatc ttcgacatgc tgctggctac atcatctcgg ttccgcatga
     3481 tgaatctgca gggagaggag tttgtgtgcc tcaaatctat tattttgctt aattctggag
     3541 tgtacacatt tctgtccagc accctgaagt ctctggaaga gaaggaccat atccaccgag
     3601 tcctggacaa gatcacagac actttgatcc acctgatggc caaggcaggc ctgaccctgc
     3661 agcagcagca ccagcggctg gcccagctcc tcctcatcct ctcccacatc aggcacatga
     3721 gtaacaaagg catggagcat ctgtacagca tgaagtgcaa gaacgtggtg cccctctatg
     3781 acctgctgct ggaggcggcg gacgcccacc gcctacatgc gcccactagc cgtggagggg
     3841 catccgtgga ggagacggac caaagccact tggccactgc gggctctact tcatcgcatt
     3901 ccttgcaaaa gtattacatc acgggggagg cagagggttt ccctgccaca gcttaagata
     3961 tcactcctca ggtgcaggct gcctatcaga aggtggtggc tggtgtggcc aatgccctgg
     4021 ctcacaaata ccactgagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc
     4081 cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt
     4141 ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc
     4201 agaatgagca tttggtttag agtttggcaa catatgccca tatgctggct gccatgaaca
     4261 aaggttggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat
     4321 tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt
     4381 tttctttaac atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct
     4441 cctgactact cccagtcata gctgtccctc ttctcttatg aagatctatt ctatagtgtc
     4501 acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4561 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4621 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4681 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4741 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4801 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4861 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4921 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4981 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5041 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5101 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5161 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5221 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5281 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5341 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5401 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5461 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5521 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5581 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5641 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5701 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5761 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5821 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5881 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5941 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6001 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6061 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6121 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6181 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6241 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6301 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6361 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6421 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6481 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6541 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6601 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6661 cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     6721 cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     6781 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     6841 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     6901 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     6961 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     7021 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     7081 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     7141 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag
     7201 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     7261 gaaaggggga tgtgctgcaa ggcgattaag ttggg
//
