LOCUS       sequence-318375-        6901 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Constitutive expression of 3xFLAG-NLS-DsDed-ZF10 under the EF1-alpha
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6901)
  AUTHORS   Muldoon JJ, Kandula V, Hong M, Donahue PS, Boucher JD, Bagheri N, 
            Leonard JN
  TITLE     Model-guided design of mammalian genetic programs.
  JOURNAL   Sci Adv. 2021 Feb 19;7(8). pii: 7/8/eabe9375. doi: 
            10.1126/sciadv.abe9375. Print 2021 Feb.
  PUBMED    33608279
REFERENCE   2  (bases 1 to 6901)
  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: "Sci Adv."; 
            date: "2021-02-19"; volume: "7(8). pii: 7/8/eabe9375. doi"; pages: "
            10.1126/sciadv.abe9375. Print 2021 Feb"
COMMENT     Sequence Label: pJM507 EF1-alpha 3xFLAG-NLS-DsDed-ZF10 in TUPV2
FEATURES             Location/Qualifiers
     source          1..6901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          231..1173
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1130..1150
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1184..1208
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1283..1292
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1292..1357
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1364..1384
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1427..2101
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWEKVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(1607..1627)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2019..2042
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2458..2475)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2464..2688
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3457..3885
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3544..3563)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3754..3775
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3908..4104
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3955..4090
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4017..4036
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4224..4345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4261..4280)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4315..4334
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4394..4410)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4394..4410)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4407..4429)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4418..4434
                     /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(4442..4448,4449..4466,4467..4472))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4487..4508
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4625..4642)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4796..5384)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4876..4895)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5555..6346,6347..6415))
                     /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     6178..6197
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6416..6520)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6901,1..1182)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
ORIGIN
        1 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
       61 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
      121 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
      181 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
      241 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
      301 cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
      361 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
      421 tggcttgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
      481 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
      541 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
      601 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
      661 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
      721 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
      781 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
      841 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
      901 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
      961 gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     1021 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     1081 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     1141 acagtggttc aaagtttttt tcttccattt caggtgtcgt gagagctcgt ttagtgaacc
     1201 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga gaacgggacc
     1261 gatccaaatt ctcgaggcta gcgccaccat ggattataag gatcacgatg gcgactacaa
     1321 ggaccacgat attgactaca aagacgatga cgataaaatg gctcctaaga aaaagcgcaa
     1381 agtcggtatc catggcgttc ccggatcagg ctccggaagt ggctccatgg atagcactga
     1441 gaacgtcatc aagcccttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     1501 cgagttcgag atcgagggcg agggcgaggg caagccctac gagggcaccc agaccgccaa
     1561 gctccaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt
     1621 ccagtacggc tccaaggtgt acgtgaagca ccccgccgac atccccgact acaagaagct
     1681 gtccttcccc gagggcttca agtgggagaa ggtgatgaac ttcgaggacg gcggcgtggt
     1741 gaccgtgacc caggactcct ccctccagga cggcaccttc atctaccacg tgaagttcat
     1801 cggcgtgaac ttcccctccg acggccccgt aatgcagaag aaaactctgg gctgggagcc
     1861 ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc acaaggcgct
     1921 gaagctgaag ggcggcggcc actacctggt ggagttcaag tcaatctaca tggccaagaa
     1981 gcccgtgaag ctgcccggct actactacgt ggactccaag ctggacatca cctcccacaa
     2041 cgaggactac accgtggtgg agcagtacga gcgcgccgag gcccgccacc acctgttcca
     2101 gtctggttct ggttctggtt caggatccgg taccggtgga gctagacccg gggagcgccc
     2161 cttccagtgt cgcatttgca tgcggaactt ttcgactaat caaaaattgg aagttcatac
     2221 ccgtactcat accggtgaaa aaccgtttca gtgtcggatc tgtatgcgaa atttctccgt
     2281 tagacataat ttgcaaagac atctacgtac gcacaccggc gagaagccat tccaatgccg
     2341 aatatgcatg cgcaacttca gtcaacatcc aaatttgact agacacctaa aaacccacct
     2401 gagaggatct taagcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct
     2461 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     2521 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     2581 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     2641 attgggaagt caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcag
     2701 gtcgaagaac tatttcctgg ctgttacgcg agcgcatcgc gataagctct agagggacag
     2761 ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg
     2821 agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg
     2881 cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg
     2941 ctgctctttg agcctgcaga cacctggggg gatacgggga aaaagcttta ggctgaaaga
     3001 gagatttaga atgacagaat catagaacgg cctgcgtacg gatggggctc tagctctaga
     3061 gcttgatatc gaattcctgc agccccgcta gagggacagc ccccccccaa agcccccagg
     3121 gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc
     3181 ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg
     3241 gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac
     3301 acctgggggg atacggggaa aaagctttag gctgaaagag agatttagaa tgacagaatc
     3361 atagaacggc ctgggcgcgc ctggtggcga acgatcaaca gacgggagag gactaaagtc
     3421 ttcgctgaca gcgaccggac tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3481 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3541 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3601 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     3661 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     3721 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     3781 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     3841 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     3901 gtgtcagtta ggctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     3961 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4021 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     4081 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggaggt
     4141 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4201 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4261 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4321 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4381 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4441 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4501 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4561 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4621 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4681 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4741 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4801 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4861 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4921 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4981 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5041 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5101 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5161 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5221 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5281 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5341 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5401 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5461 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5521 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5581 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5641 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagatc
     5701 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5761 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5821 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5881 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5941 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6001 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6061 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6121 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6181 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6241 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6301 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6361 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6421 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6481 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6541 cacctgacgt cgacggatcg ggagatcgaa gacgggcaaa accgatgttg acggactaat
     6601 cctgacaaga tctggggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     6661 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     6721 tcgcgatgta cgggccagat atacgcgtga gtaattcata caaaaggact cgcccctgcc
     6781 ttggggaatc ccagggaccg tcgttaaact cccactaacg tagaacccag agatcgctgc
     6841 gttcccgccc cctcacccgc ccgctctcgt catcactgag gtggagaaga gcatgcgtga
     6901 g
//
