LOCUS       Exported                6199 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Inducible superfolder GFP (sfGFP) expression, controlled under T7 
            bacteriophage promoter.
ACCESSION   .
VERSION     .
KEYWORDS    DT042A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6199)
  AUTHORS   Toparlak OD, Zasso J, Bridi S, Serra MD, Macchi P, Conti L, Baudet 
            ML, Mansy SS
  TITLE     Artificial cells drive neural differentiation.
  JOURNAL   Sci Adv. 2020 Sep 18;6(38). pii: 6/38/eabb4920. doi: 
            10.1126/sciadv.abb4920. Print 2020 Sep.
  PUBMED    32948587
REFERENCE   2  (bases 1 to 6199)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6199
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(923..2005)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1967..1986
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2006..2083)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2289..2308
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    2415..2431
                     /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)."
     protein_bind    2479..2503
                     /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)."
     RBS             2518..2540
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2549..2566
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2579..3292
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      3421..3468
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3490..3945
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3577..3596)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3787..3808
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3971..4075
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4076..4936
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4294..4313)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5107..5695
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5596..5615
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5849..5866
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5881..6020
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5946..5965)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6106..6128
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6122..114)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
       61 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
      121 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
      181 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
      241 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
      301 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
      361 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
      421 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
      481 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
      541 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
      601 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
      661 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
      721 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
      781 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
      841 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
      901 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
      961 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     1021 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     1081 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     1141 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     1201 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     1261 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     1321 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     1381 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     1441 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     1501 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     1561 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     1621 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     1681 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     1741 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     1801 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     1861 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     1921 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     1981 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     2041 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     2101 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     2161 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     2221 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     2281 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     2341 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa tgaaatcata
     2401 aaaaattatt tgctttgtga gcggataaca attataatag attcaagccc ggatagctca
     2461 gtcggtagag cagcggccgg aattgtgagc ggataacaat tcccctctag aaataatttt
     2521 gtttaacttt aagaaggaga tatacatgca tcatcaccac catcatggcg gtggctcgat
     2581 gcgtaaaggc gaagagctgt tcactggtgt cgtccctatt ctggtggaac tggatggtga
     2641 tgtcaacggt cataagtttt ccgtgcgtgg cgagggtgaa ggtgacgcaa ctaatggtaa
     2701 actgacgctg aagttcatct gtactactgg taaactgccg gtaccttggc cgactctggt
     2761 aacgacgctg acttatggtg ttcagtgctt tgctcgttat ccggaccata tgaagcagca
     2821 tgacttcttc aagtccgcca tgccggaagg ctatgtgcag gaacgcacga tttcctttaa
     2881 ggatgacggc acgtacaaaa cgcgtgcgga agtgaaattt gaaggcgata ccctggtaaa
     2941 ccgcattgag ctgaaaggca ttgactttaa agaagacggc aatatcctgg gccataagct
     3001 ggaatacaat tttaacagcc acaatgttta catcaccgcc gataaacaaa aaaatggcat
     3061 taaagcgaat tttaaaattc gccacaacgt ggaggatggc agcgtgcagc tggctgatca
     3121 ctaccagcaa aacactccaa tcggtgatgg tcctgttctg ctgccagaca atcactatct
     3181 gagcacgcaa agcgttctgt ctaaagatcc gaacgagaaa cgcgatcata tggttctgct
     3241 ggagttcgta accgcagcgg gcatcacgca tggtatggat gaactgtaca aataataacg
     3301 actcaggctg ctacgcctgt gtactggaaa acaaaaccaa aacccaaaaa acaaaaaact
     3361 gagcccattg gtatcgtgga aggactcgat caaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3421 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgat atccggattg
     3481 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3541 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3601 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3661 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3721 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3781 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3841 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3901 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     3961 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     4021 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     4081 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     4141 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     4201 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4261 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4321 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4381 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4441 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4501 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4561 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     4621 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4681 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4741 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4801 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4861 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4921 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4981 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5041 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5101 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5161 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5221 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     5281 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5341 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5401 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5461 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5521 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5581 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5641 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5701 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5761 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5821 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     5881 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     5941 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     6001 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     6061 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     6121 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     6181 cgtggtcgtg aagcgattc
//
