LOCUS       Exported                5708 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    zbFGF
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5708)
  AUTHORS   Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson 
            JA
  TITLE     Feeder-independent culture of human embryonic stem cells.
  JOURNAL   Nat Methods. 2006 Aug . 3(8):637-646.
  PUBMED    16862139
REFERENCE   2  (bases 1 to 5708)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5708
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1328..1519
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1513..1535)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1621..1760
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1676..1695
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1775..1792)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1946..2534)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2026..2045)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2645..2664)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2656..3471
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2729..2748)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3563..4018)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3700..3721)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3912..3931
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      4055..4102
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4098..4116
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4169..4186)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4678..4700
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4715..4739
                     /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     complement(4739..4758)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4740..4758)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4842..4861)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5067..5144
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5145..519
                     /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     complement(5164..5183)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
       61 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
      121 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
      181 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
      241 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
      301 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
      361 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
      421 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
      481 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
      541 ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt
      601 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
      661 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg
      721 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc
      781 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat
      841 tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg
      901 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac
      961 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg
     1021 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg
     1081 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa
     1141 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg
     1201 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag
     1261 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga
     1321 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
     1381 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg
     1441 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga
     1501 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     1561 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     1621 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     1681 tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac agatgcgtaa
     1741 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1801 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1861 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1921 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1981 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2041 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2101 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2161 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2221 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2281 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2341 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     2401 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2461 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2521 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2581 aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta cataaacagt
     2641 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag gccgcgatta
     2701 aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     2761 tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt gtttctgaaa
     2821 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg
     2881 acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg
     2941 ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga atatcctgat
     3001 tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct
     3061 gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga
     3121 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     3181 gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact
     3241 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     3301 gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc
     3361 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     3421 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta agaattaatt
     3481 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     3541 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     3601 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3661 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     3721 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     3781 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     3841 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     3901 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     3961 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc
     4021 cattcgccaa tccggatata gttcctcctt tcagcaaaaa acccctcaag acccgtttag
     4081 aggccccaag gggttatgct agttattgct cagcggtggc agcagccaac tcagcttcct
     4141 ttcgggcttt gttagcagcc ggatctcagt ggtggtggtg gtggtgctcg agtgcggccg
     4201 caagcttcag catttggccg acatgggcag aaacaggatg gccttctgtc ccgggctggt
     4261 tttagagccg cttttatact ggccggttct cttcagagcc acgtaccagt cgggatactt
     4321 gcgggatctg tatgtgttgt agttgttgga ctccaggcgc tccaggaaat aacattcatc
     4381 tgtggtcctt ttcgtcccga acagtcgtcc gtctgcgttc atggcaagga aacggttggt
     4441 acagatgcct ttaatgagta cttcacccac tgccgtcgcc tgcagctgca gacgaatgtg
     4501 cgggtcgttc ttgtctcggg ctccgtccac tcggccgtcc gcgttgatcc gcaggaagaa
     4561 tcctccgttt ttgcagtaca ggcgcttggg atccctgaag ctgcccgcgg gaaagctgct
     4621 gttttcggcg tccggagcgg gcgggagtgt ggtgatccct ccggtggcca tggtatatct
     4681 ccttcttaaa gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc
     4741 ctatagtgag tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat
     4801 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
     4861 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
     4921 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
     4981 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
     5041 gcataaggga gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta
     5101 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     5161 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     5221 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     5281 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     5341 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     5401 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     5461 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     5521 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     5581 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     5641 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5701 cgggccca
//
