LOCUS       sequence-267613-        6636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses human ETV2 upon doxycycline treatment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6636)
  AUTHORS   Ream MW, Randolph LN, Jiang Y, Chang Y, Bao X, Lian XL
  TITLE     Direct programming of human pluripotent stem cells into endothelial 
            progenitors with SOX17 and FGF2.
  JOURNAL   Stem Cell Reports. 2024 Mar 5:S2213-6711(24)00044-4. doi: 
            10.1016/j.stemcr.2024.02.006.
  PUBMED    38518781
REFERENCE   2  (bases 1 to 6636)
  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: "Stem Cell 
            Reports. 2024 Mar 5:S2213-6711(24)00044-4. doi: 
            10.1016/j.stemcr.2024.02.006."
COMMENT     Sequence Label: XLone-ETV2
FEATURES             Location/Qualifiers
     source          1..6636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(397..420)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      420..429
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(449..660)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        702..1069
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    710..728
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    746..764
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    782..800
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    818..836
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    854..872
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    890..908
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    926..944
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      1076..1085
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2291..2425
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2701..2735
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2746..2763)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2746..2762)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2755..2777)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2971..2990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3090..3112
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3150..3168)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3236..3340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3341..3409,3410..4201)
                     /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(3559..3578)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4372..4960
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4861..4880
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5114..5131
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5248..5269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5284..5289,5290..5307,5308..5314)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5322..5338
                     /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     5327..5349
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5346..5362
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5346..5362
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5388..5450
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5626..5707
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5824..6222)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(6223..6279)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(join(6289..6636,1..396))
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
ORIGIN
        1 gtacagtgcg ttctccaggg agaagccttg ctgacacagg aacgcgagct gattttccag
       61 ggtttcgtac tgtttctctg ttgggcgggt gccgagatgc actttagccc cgtcgcgatg
      121 tgagaggaga gcacagcggt atgacttggc gttgttccgc agaaagtctt gccatgactc
      181 gccttccagg gggcaggagt gggtatgatg cctgtccagc atctcgattg gcagggcatc
      241 gagcagggcc cgcttgttct tcacgtgcca gtacagggta ggctgctcaa ctcccagctt
      301 ttgagcgagt ttccttgtcg tcaggccttc gataccgact ccattgagta attccagagc
      361 agagtttatg actttgctct tgtccagtct agacatctta tcgtcatcgt ctttgtaatc
      421 catggtggcg gatcccgcgt cacgacacct gtgttctggc ggcaaacccg ttgcgaaaaa
      481 gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg ggaaaaaggc
      541 ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc taggcaccgg
      601 ttcaattgcc gacccctccc cccaacttct cggggactgt gggcgatgtg cgctctgccc
      661 actgacgggc accggagcca ctcgagtgga attatcacct cgagtttact ccctatcagt
      721 gatagagaac gtatgaagag tttactccct atcagtgata gagaacgtat gcagacttta
      781 ctccctatca gtgatagaga acgtataagg agtttactcc ctatcagtga tagagaacgt
      841 atgaccagtt tactccctat cagtgataga gaacgtatct acagtttact ccctatcagt
      901 gatagagaac gtatatccag tttactccct atcagtgata gagaacgtat aagctttgct
      961 tatgtaaacc agggcgccta taaaagagtg ctgatttttt gagtaaactt caattccaca
     1021 acacttttgt cttataccaa ctttccgtac cacttcctac cctcgtaaag gtaccgccac
     1081 catggacctg tggaactggg atgaggcatc cccacaggaa gtgcctccag ggaacaagct
     1141 ggcagggctt gaaggagcca aattaggctt ctgtttccct gatctggcac tccaagggga
     1201 cacgccgaca gcgacagcag agacatgctg gaaaggtaca agctcatccc tggcaagctt
     1261 cccacagctg gactggggct ccgcgttact gcacccagaa gttccatggg gggcggagcc
     1321 cgactctcag gctcttccgt ggtccgggga ctggacagac atggcgtgca cagcctggga
     1381 ctcttggagc ggcgcctcgc agaccctggg ccccgcccct ctcggcccgg gccccatccc
     1441 cgccgccggc tccgaaggcg ccgcgggcca gaactgcgtc cccgtggcgg gagaggccac
     1501 ctcgtggtcg cgcgcccagg ccgccgggag caacaccagc tgggactgtt ctgtggggcc
     1561 cgacggcgat acctactggg gcagtggcct gggcggggag ccgcgcacgg actgtaccat
     1621 ttcgtggggc gggcccgcgg gcccggactg taccacctcc tggaacccgg ggctgcatgc
     1681 gggtggcacc acctctttga agcggtacca gagctcagct ctcaccgttt gctccgaacc
     1741 gagcccgcag tcggaccgtg ccagtttggc tcgatgcccc aaaactaacc accgaggtcc
     1801 cattcagctg tggcagttcc tcctggagct gctccacgac ggggcgcgta gcagctgcat
     1861 ccgttggact ggcaacagcc gcgagttcca gctgtgcgac cccaaagagg tggctcggct
     1921 gtggggcgag cgcaagagaa agccgggcat gaattacgag aagctgagcc ggggccttcg
     1981 ctactactat cgccgcgaca tcgtgcgcaa gagcgggggg cgaaagtaca cgtaccgctt
     2041 cgggggccgc gtgcccagcc tagcctatcc ggactgtgcg ggaggcggac ggggagcaga
     2101 gacacaataa actagtagac cacctcccct gcgagctaag ctggacagcc aatgacgggt
     2161 aagagagtga catttttcac taacctaaga caggagggcc gtcagagcta ctgcctaatc
     2221 caaagacggg taaaagtgat aaaaatgtat cactccaacc taagacaggc gcagcttccg
     2281 agggatttga gatccagaca tgataagata cattgatgag tttggacaaa ccaaaactag
     2341 aatgcagtga aaaaaatgcc ttatttgtga aatttgtgat gctattgcct tatttgtaac
     2401 cattataagc tgcaataaac aagtttgata tctataacaa gaaaatatat atataataag
     2461 ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta aatcttaaaa
     2521 gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca
     2581 gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg actgagatgt
     2641 cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat ttcaagaatg
     2701 catgcgtcaa ttttacgcag actatctttc tagggttaag aattcactgg ccgtcgtttt
     2761 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     2821 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     2881 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2941 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3001 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3061 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3121 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     3181 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     3241 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3301 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3361 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3421 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3481 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3541 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3601 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3661 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3721 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3781 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3841 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3901 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3961 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4021 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4081 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4141 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4201 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4261 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4321 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4381 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4441 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4501 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4561 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4621 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4681 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4741 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4801 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4861 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4921 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4981 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     5041 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5101 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     5161 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     5221 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     5281 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     5341 tcacacagga aacagctatg accatgatta cgccaaggtc gacttaaccc tagaaagata
     5401 atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg tgttttatcg
     5461 gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat tatatttaca
     5521 cttacatact aataataaat tcaacaaaca atttatttat gtttatttat ttattaaaaa
     5581 aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttagcagt gaaaaaaatg
     5641 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     5701 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     5761 ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg atcctctgga
     5821 gatttagccc tcccacacat aaccagaggg cagcaattca cgaatcccaa ctgccgtcgg
     5881 ctgtccatca ctgtccttca ctatggcttt gatcccagga tgcagatcga gaagcacctg
     5941 tcggcaccgt ccgcaggggc tcaagatgcc cctgttctca tttccgatcg cgacgataca
     6001 agtcaggttg ccagctgccg cagcagcagc agtgcccagc accacgagtt ctgcacaagg
     6061 tcccccagta aaatgatata cattgacacc agtgaagatg cggccgtcgc tagagagagc
     6121 tgcgctggcg acgctgtagt cttcagagat ggggatgctg ttgattgtag ccgttgctct
     6181 ttcaatgagg gtggattctt cttgagacaa aggcttggcc atggggccgg ggttctcctc
     6241 cacgtcgccg gcctgcttca gcaggctgaa gttggtggcg ccgctgcccc cggggagcat
     6301 gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata tcaaggtcaa agtcgtcaag
     6361 ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg gcgtcggtcg gcccgccgct
     6421 ttcgcacttt agctgtttct ccaggccaca tatgattagt tccaggccga aaaggaaggc
     6481 aggttcggct ccctgccggt cgaacagctc aattgcttgt ttcagaagtg ggggcataga
     6541 atcggtggta ggtgtctctc tttcctcttt tgctacttga tgctcctgtt cctccaatac
     6601 gcagcccagt gtaaagtggc ccacggcgga cagagc
//
