LOCUS       sequence-354944-        6419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable and temporal expression control of SPI1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6419)
  AUTHORS   Jung J, Chang Y, Jin G, Lian X, Bao X
  TITLE     Temporal Expression of Transcription Factor ID2 Improves Natural 
            Killer Cell Differentiation from Human Pluripotent Stem Cells.
  JOURNAL   ACS Synth Biol. 2022 May 24. doi: 10.1021/acssynbio.2c00017.
  PUBMED    35608547
REFERENCE   2  (bases 1 to 6419)
  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: "ACS Synth 
            Biol. 2022 May 24. doi: 10.1021/acssynbio.2c00017."
COMMENT     Sequence Label: XLone-BSD SPI1
FEATURES             Location/Qualifiers
     source          1..6419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1017..1079
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    1255..1336
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1453..1851)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(1852..1908)
                     /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(1918..2661)
                     /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"
     CDS             complement(2662..2685)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2685..2694
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2714..2925)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        2967..3334
                     /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    2975..2993
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3011..3029
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3047..3065
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3083..3101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3119..3137
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3155..3173
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3191..3209
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     polyA_signal    4339..4473
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   4749..4783
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4794..4811)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4794..4810)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4803..4825)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5019..5038)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5138..5160
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5198..5216)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5284..5388
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5389..5457,5458..6249)
                     /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(5607..5626)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaaggtcg acttaaccct
     1021 agaaagataa tcatattgtg acgtacgtta aagataatca tgcgtaaaat tgacgcatgt
     1081 gttttatcgg tctgtatatc gaggtttatt tattaatttg aatagatatt aagttttatt
     1141 atatttacac ttacatacta ataataaatt caacaaacaa tttatttatg tttatttatt
     1201 tattaaaaaa aaacaaaaac tcaaaatttc ttctataaag taacaaaact tttagcagtg
     1261 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     1321 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     1381 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga
     1441 tcctctggag atttagccct cccacacata accagagggc agcaattcac gaatcccaac
     1501 tgccgtcggc tgtccatcac tgtccttcac tatggctttg atcccaggat gcagatcgag
     1561 aagcacctgt cggcaccgtc cgcaggggct caagatgccc ctgttctcat ttccgatcgc
     1621 gacgatacaa gtcaggttgc cagctgccgc agcagcagca gtgcccagca ccacgagttc
     1681 tgcacaaggt cccccagtaa aatgatatac attgacacca gtgaagatgc ggccgtcgct
     1741 agagagagct gcgctggcga cgctgtagtc ttcagagatg gggatgctgt tgattgtagc
     1801 cgttgctctt tcaatgaggg tggattcttc ttgagacaaa ggcttggcca tggggccggg
     1861 gttctcctcc acgtcgccgg cctgcttcag caggctgaag ttggtggcgc cgctgccccc
     1921 ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa
     1981 gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggtcgg
     2041 cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt ccaggccgaa
     2101 aaggaaggca ggttcggctc cctgccggtc gaacagctca attgcttgtt tcagaagtgg
     2161 gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat gctcctgttc
     2221 ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca gtgcgttctc
     2281 cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt cgtactgttt
     2341 ctctgttggg cgggtgccga gatgcacttt agccccgtcg cgatgtgaga ggagagcaca
     2401 gcggtatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt ccagggggca
     2461 ggagtgggta tgatgcctgt ccagcatctc gattggcagg gcatcgagca gggcccgctt
     2521 gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag cgagtttcct
     2581 tgtcgtcagg ccttcgatac cgactccatt gagtaattcc agagcagagt ttatgacttt
     2641 gctcttgtcc agtctagaca tcttatcgtc atcgtctttg taatccatgg tggcggatcc
     2701 cgcgtcacga cacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga
     2761 ctactgcact tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga
     2821 catcactttc ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc
     2881 ctccccccaa cttctcgggg actgtgggcg atgtgcgctc tgcccactga cgggcaccgg
     2941 agccactcga gtggaattat cacctcgagt ttactcccta tcagtgatag agaacgtatg
     3001 aagagtttac tccctatcag tgatagagaa cgtatgcaga ctttactccc tatcagtgat
     3061 agagaacgta taaggagttt actccctatc agtgatagag aacgtatgac cagtttactc
     3121 cctatcagtg atagagaacg tatctacagt ttactcccta tcagtgatag agaacgtata
     3181 tccagtttac tccctatcag tgatagagaa cgtataagct ttgcttatgt aaaccagggc
     3241 gcctataaaa gagtgctgat tttttgagta aacttcaatt ccacaacact tttgtcttat
     3301 accaactttc cgtaccactt cctaccctcg taaaggtacc gccaccatgt tacaggcgtg
     3361 caaaatggaa gggtttcccc tcgtcccccc tccatcagaa gacctggtgc cctatgacac
     3421 ggatctatac caacgccaaa cgcacgagta ttacccctat ctcagcagtg atggggagag
     3481 ccatagcgac cattactggg acttccaccc ccaccacgtg cacagcgagt tcgagagctt
     3541 cgccgagaac aacttcacgg agctccagag cgtgcagccc ccgcagctgc agcagctcta
     3601 ccgccacatg gagctggagc agatgcacgt cctcgatacc cccatggtgc caccccatcc
     3661 cagtcttggc caccaggtct cctacctgcc ccggatgtgc ctccagtacc catccctgtc
     3721 cccagcccag cccagctcag atgaggagga gggcgagcgg cagagccccc cactggaggt
     3781 gtctgacggc gaggcggatg gcctggagcc cgggcctggg ctcctgcctg gggagacagg
     3841 cagcaagaag aagatccgcc tgtaccagtt cctgttggac ctgctccgca gcggcgacat
     3901 gaaggacagc atctggtggg tggacaagga caagggcacc ttccagttct cgtccaagca
     3961 caaggaggcg ctggcgcacc gctggggcat ccagaagggc aaccgcaaga agatgaccta
     4021 ccagaagatg gcgcgcgcgc tgcgcaacta cggcaagacg ggcgaggtca agaaggtgaa
     4081 gaagaagctc acctaccagt tcagcggcga agtgctgggc cgcaggggcc tggccgagcg
     4141 gcgccacccg ccccactgac tagtagacca cctcccctgc gagctaagct ggacagccaa
     4201 tgacgggtaa gagagtgaca tttttcacta acctaagaca ggagggccgt cagagctact
     4261 gcctaatcca aagacgggta aaagtgataa aaatgtatca ctccaaccta agacaggcgc
     4321 agcttccgag ggatttgaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     4381 aaaactagaa tgcagtgaaa aaaatgcctt atttgtgaaa tttgtgatgc tattgcctta
     4441 tttgtaacca ttataagctg caataaacaa gtttgatatc tataacaaga aaatatatat
     4501 ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt atgagttaaa
     4561 tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt cgttatagtt
     4621 caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc aagcggcgac
     4681 tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag agcaatattt
     4741 caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaagaa ttcactggcc
     4801 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     4861 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     4921 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat
     4981 ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca
     5041 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5101 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5161 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     5221 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     5281 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5341 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5401 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5461 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5521 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5581 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5641 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5701 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5761 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5821 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5881 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5941 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     6001 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     6061 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     6121 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6181 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6241 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6301 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6361 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
